Genetic markers for engraftment of human cardiac ventricular progenitor cells

ABSTRACT

The present invention provides genetic markers for identifying engraftable human cardiac ventricular progenitor cells. The engraftment markers of the invention include angiogenic markers and extracellular matrix markers. Human ventricular progenitor cells expressing these markers are capable of forming ventricular tissue in vivo that is vascularized and supported by an extracellular matrix. Methods of engrafting human cardiac ventricular progenitor cells by transplanting into a subject progenitor cells that express the engraftment markers are also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to, and the benefit of, U.S.Application No. 62/297,217, filed on Feb. 19, 2016, the contents ofwhich are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Heart failure, predominantly caused by myocardial infarction, is theleading cause of death in both adults and children worldwide and isincreasing exponentially worldwide (Bui, A. L. et al. (2011) Nat. Rev.Cardiol. 8:30-41). The disease is primarily driven by the loss ofventricular muscle that occurs during myocardial injury (Lin, Z. and Pu,W. T. (2014) Sci. Transl. Med. 6:239rv1) and is compounded by thenegligible ability of the adult heart to mount a regenerative response(Bergmann, O. et al. (2009) Science 324:98-102; Senyo, S. E. et al.(2013) Nature 493:433-436). Although heart transplantation can becurative, the markedly limited availability of human heart organ donorshas led to a widespread unmet clinical need for a renewable source ofpure, mature and functional human ventricular muscle tissue (Segers, V.F. M. and Lee, R. J. (2008) Nature 451:937-942; Später, D. et al. (2014)Development 141:4418-4431).

Human pluripotent stem cells (hPSCs) offer the potential to generatelarge numbers of functional cardiomyocytes for potential clinicalrestoration of function in damaged or diseased hearts. Transplantationof stem cells into the heart to improve cardiac function and/or toenrich and regenerate damaged myocardium has been proposed (see e.g.,U.S. Patent Publication 20040180043). Combination therapy, in whichadult stem cells are administered in combination with treatment withgrowth factor proteins has also been proposed (see e.g., U.S. PatentPublication 20050214260).

A significant advancement in the approach of cell transplantation toimprove cardiac function and regenerate heart tissue was theidentification and isolation of a family of multipotent cardiacprogenitor cells that are capable of giving rise to cardiac myocytes,cardiac smooth muscle and cardiac endothelial cells (Cal, C. L. et al.(2003) Dev. Cell. 5:877-889; Moretti, A. et al. (2006) Cell127:1151-1165; Bu, L. et al. (2009) Nature 460:113-117; U.S. PatentPublication 20060246446). These cardiac progenitor cells arecharacterized by the expression of the LIM homeodomain transcriptionfactor Islet 1 (Isl1) and thus are referred to as Isl1+ cardiacprogenitor cells. (Ibid). In contrast, Isl1 is not expressed indifferentiated cardiac cells. Additional markers of the Isl1+ cardiacprogenitor cells that arise later in differentiation than Isl1 have beendescribed and include Nkx2.5 and flk1 (see e.g., U.S. Patent Publication20100166714).

The renewal and differentiation of Isl1+ cardiac progenitor cells hasbeen shown to be regulated by a Wnt/beta-catenin signaling pathway (seee.g., Qyang, Y. et al. (2007) Cell Stem Cell. 1:165-179; Kwon, C. et al.(2007) Proc. Natl. Acad. Sci. USA 104:10894-10899). This has led to thedevelopment of methods to induce a pluripotent stem cell to enter theIsl1+ lineage and for expansion of the Isl1+ population throughmodulation of Wnt signaling (see e.g., Lian, X. et al. (2012) Proc.Natl. Acad. Sci. USA 109:E1848-57; Lian, X. et al. (2013) Nat. Protoc.8:162-175; U.S. Patent Publication 20110033430; U.S. Patent Publication20130189785).

While human pluripotent stem cells hold great promise, a significantchallenge has been the ability to move from simply differentiation ofdiverse cardiac cells to forming a larger scale pure 3D ventricularmuscle tissue in vivo, which ultimately requires vascularization,assembly and alignment of an extracellular matrix, and maturation.Toward that end, a diverse population of cardiac cells (atrial,ventricular, pacemaker) has been coupled with artificial anddecellurized matrices (Masumoto, H. et al. (2014) Sci. Rep. 4:5716; Ott,H. C. et al. (2008) Nat. Med. 14:213-221; Schaaf, S. et al. (2011) PLoSOne 6:e26397), vascular cells and conduits (Tulloch, N. L. et al. (2011)Circ. Res. 109:47-59) and cocktails of microRNAs (Gama-Garvalho, M. etal. (2014) Cells 3:996-1026) have been studied, but the goal remainselusive.

Accordingly, there is still a need in the art for genetics markers ofcardiac progenitor cells that would allow for identification of thosecells that can successfully form a tissue graft in vivo, in particulargenetic markers for identification of progenitor cells that candifferentiate into ventricular muscle cells, as well as achievevascularization and extracellular matrix formation in vivo.

SUMMARY OF THE INVENTION

This invention provides genetic markers, referred to as engraftmentmarkers, that identify human ventricular progenitor cells (HVPs) whichhave the capacity to engraft in vivo. Transplantation of the HVPs invivo generates a graft of mature ventricular muscle tissue that isvascularized and has an extracellular matrix. HVPs have previously beenshown to express cell surface markers such as JAG1, FZD4, LIFR, FGFR3and/or TNFSF9, as well as the intracellular marker Islet 1 (see U.S.Ser. No. 14/832,324, filed Aug. 21, 2015, and U.S. Ser. No. 14/984,783,filed Dec. 30, 2015, the entire contents of each of which are expresslyincorporated herein by reference). The engraftment markers of theinvention now allow for the identification of cells within thatpopulation which have the capacity for engraftment. In one embodiment,the engraftment marker is an angiogenic marker. In another embodiment,the engraftment marker is an extracellular matrix marker. The inventionprovides both “positive” markers, whose expression strongly correlateswith the expression of other HVP markers to thereby allow foridentification of engraftable HVPs, as well as “negative” markers, whoselack of expression strongly correlates with the expression of other HVPmarkers to thereby also allow for identification of engraftable HVPs.

Accordingly, in one aspect, the invention provides a method ofidentifying engraftable human ventricular progenitor cells (HVPs), themethod comprising:

-   -   detecting expression of at least one engraftment marker in the        HVPs to thereby identify engraftable HVPs, wherein:    -   the HVPs also express at least one surface marker selected from        the group consisting of: JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.

In another aspect, the invention provides a method of isolatingengraftable human ventricular progenitor cells (HVPs), the methodcomprising:

-   -   culturing human cells containing cardiac progenitor cells under        conditions causing differentiation into human ventricular        progenitor cells (HVPs);    -   detecting expression on the HVPs of at least one surface marker        selected from the group consisting of JAG1, FZD4, LIFR, FGFR3        and/or TNFSF9;    -   detecting expression in the HVPs of at least one engraftment        marker; and    -   isolating HVPs that co-express the at least one surface marker        and the at least one engraftment marker to thereby isolate        engraftable HVPs.

In another aspect, the invention provides a method for engrafting humanventricular tissue in a subject, the method comprising:

-   -   transplanting engraftable human ventricular progenitor cells        (HVPs) into the subject such that human ventricular tissue is        engrafted in the subject,    -   wherein prior to transplantation, engraftable HVPs are        identified by detecting expression of at least one engraftment        marker in the HVPs; and    -   wherein the HVPs express at least one surface marker selected        from the group consisting of: JAG1, FZD4, LIFR, FGFR3 and/or        TNFSF9.

In another aspect, the invention provides a method for engrafting humanventricular tissue in a subject, the method comprising:

-   -   culturing human cells containing cardiac progenitor cells under        conditions causing differentiation into human ventricular        progenitor cells (HVPs);    -   detecting expression on the HVPs of at least one surface marker        selected from the group consisting of JAG1, FZD4, LIFR, FGFR3        and/or TNFSF9;    -   detecting expression in the HVPs of at least one engraftment        marker;    -   isolating HVPs that co-express the at least one surface marker        and the at least one engraftment marker to thereby isolate        engraftable HVPs; and    -   transplanting engraftable the HVPs into the subject such that        human ventricular tissue is engrafted in the subject.

In another aspect, the invention provides a method for engrafting humanventricular tissue in a subject, the method comprising:

-   -   culturing human cells containing cardiac progenitor cells under        conditions causing differentiation into human ventricular        progenitor cells (HVPs) expressing at least one surface marker        selected from the group consisting of JAG1, FZD4, LIFR, FGFR3        and/or TNFSF9;    -   isolating the HVPs expressing the at least one surface marker to        form an HVP population;    -   detecting expression of at least one engraftment marker in a        sample of the HVP population; and    -   transplanting the HVPs expressing the at least one surface        marker and the at least one engraftment marker into the subject        such that human ventricular tissue is engrafted in the subject.

In one embodiment, the at least one engraftment marker detected is atleast one positive angiogenic marker. More than one positive angiogenicmarker can be detected, for example, three or more, or ten or more,positive angiogenic markers can be detected.

In another embodiment, the at least one engraftment marker detected isat least one positive extracellular matrix marker. More than onepositive extracellular matrix marker can be detected, for example, threeor more, or ten or more, positive extracellular matrix markers can bedetected.

In another embodiment, the at least one engraftment marker detected isat least one positive angiogenic marker and at least one positiveextracellular matrix marker. More than one of each positive marker(angiogenic and extracellular matrix) can be detected, for example,three or more, or ten or more, of each of these positive markers can bedetected.

In one embodiment, the at least one positive angiogenic marker isselected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA,EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1,ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT,NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1,HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1,NRXN3, FZD5 and HHEX. In another embodiment, the at least one positiveangiogenic marker is selected from the group consisting of: FGF10,PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1,MEIS1, TBX1, PKNOX1, ID1, TCF21 and HEY1. In another embodiment, the atleast positive angiogenic marker is selected from the group consistingof: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2 and NTRK1.

In one embodiment, the at least one positive extracellular matrix markeris selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6,ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1,EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4,EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1,LTBP4 and FREM1. In another embodiment, the at least one positiveextracellular matrix marker is selected from the group consisting of:FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7,FBLN2, NDNF and HTRA1. In another embodiment, the at least one positiveextracellular matrix marker is selected from the group consisting of:FGF10, SMOC1 and CCBE1.

In those embodiments in which both at least one positive angiogenicmarker and at least one positive extracellular matrix marker aredetected, the at least one positive angiogenic marker can be selectedfrom the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2,NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21,HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN,SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2,CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5and HHEX; and

the at least one positive extracellular matrix marker can be selectedfrom the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12,COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1,SPOCK3, PODNL1, IHH, ALAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1,HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 andFREM1.

In another embodiment, the at least one positive angiogenic marker isselected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA,EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1,ID1, TCF21 and HEY1; and

the at least one positive extracellular matrix marker is selected fromthe group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1,LAMA1, BMP4, FBLN7, FBLN2, NDNF and HTRA1.

In another embodiment, the at least one positive angiogenic marker isselected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA,EPHB2, GATA2 and NTRK1; and

the at least one positive extracellular matrix marker is selected fromthe group consisting of: FGF10, SMOC1 and CCBE1.

In one embodiment, the at least one engraftment marker detected is atleast one negative angiogenic marker. More than one negative angiogenicmarker can be detected, for example, three or more, or ten or more,negative angiogenic markers can be detected.

In another embodiment, the at least one engraftment marker detected isat least one negative extracellular matrix marker. More than onenegative extracellular matrix marker can be detected, for example, threeor more, or ten or more, negative extracellular matrix markers can bedetected.

In another embodiment, the at least one engraftment marker detected isat least one negative angiogenic marker and at least one negativeextracellular matrix marker. More than one of each negative marker(angiogenic and extracellular matrix) can be detected, for example,three or more, or ten or more, of each of these negative markers can bedetected.

In one embodiment, the at least one negative angiogenic marker isselected from the group consisting of: ETS1, BAX, XBP1, TDGF1, C5AR1,EPHA1, HS6ST1, SHC1, SP100, JAM3, CASP8, FLT4, SFRP2, HPSE, BAK1, GPX1,VAV3, VAV2, EGF, ADAM15 and AGGF1. In another embodiment, the at leastone negative angiogenic marker is selected from the group consisting of:VAV3, VAV2, EGF, ADAM15 and AGGF1.

In one embodiment, the at least one negative extracellular matrix markeris selected from the group consisting of: FKBP1A, CLU, TFP12, PLSCR1,FBLN5, VWA1, ADAMTS16, MMP25, SFRP2 and SOD1.

In those embodiments in which both at least one negative angiogenicmarker and at least one negative extracellular matrix marker aredetected, the at least one negative angiogenic marker can be selectedfrom the group consisting of: ETS1, BAX, XBP1, TDGF1, C5AR1, EPHA1,HS6ST1, SHC1, SP100, JAM3, CASP8, FLT4, SFRP2, HPSE, BAK1, GPX1, VAV3,VAV2, EGF, ADAM15 and AGGF1; and

the at least one negative extracellular matrix marker can be selectedfrom the group consisting of: FKBP1A, CLU, TFP12, PLSCR1, FBLN5, VWA1,ADAMTS16, MMP25, SFRP2 and SOD1.

In one embodiment, detecting expression of at least one engraftmentmarker comprises detecting expression of mRNA encoding the at least oneengraftment marker in the HVPs (detection of “positive” markerexpression, such as a positive angiogenic marker(s) and/or a positiveextracellular matrix marker(s)). In another embodiment, detectingexpression of at least one engraftment marker comprises detecting lackof expression of mRNA encoding the at least one engraftment marker inthe HVPs (detection of “negative” marker expression, such as a negativeangiogenic marker(s) and/or a negative extracellular matrix marker(s)).

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWING

The drawing is a schematic diagram of an exemplary culturing protocolfor generating human Isl1+ cardiomyogenic progenitor cells from humanpluripotent stem cells (hPSCs).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of identifying engraftable humanventricular progenitor cells (HVPs), as well as methods of transplantingengraftable HVPs. The methods of the invention are based, at least inpart, on the discovery of the gene expression profile within those HVPshaving the capacity, when transplanted in vivo, to form a graft ofmature human ventricular tissue that is vascularized and has anextracellular matrix. Accordingly, detection of one or more engraftmentmarkers in HVPs prior to transplantation allows for identification ofthose cells capable of engraftment in vivo, thereby greatly increasingthe likelihood of success of the grafting process.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the term “engraftable” refers to a property of cells(such as a human ventricular progenitor cells) relating to the abilityto form a graft in vivo when transplanted into a host. Within a mixedpopulation of cells (e.g., progenitor cells), not all cells within thatmixed population will necessarily form a graft when transplanted into ahost, since there are a number of biological processes that must occurfor there to be successful engraftment. These biological processesinclude the formation of blood vessels into the graft(neovascularization) and the formation of an extracellular matrix tosupport the graft. Thus, the “engraftable” cells within a mixedpopulation of cells refers to those cells having the capacity to form agraft in vivo when transplanted into a host.

As used herein, the term “engraftment marker” refers to a marker, suchas a genetic or protein marker, detectable within cells that correlateswith the engraftability of the cells, i.e., the ability of the cells toform a graft in vivo when transplanted into a host.

As used herein, an engraftment marker can be a “positive marker”, whichrefers to those markers whose expression positively correlates withother identifying markers of human ventricular progenitor cells (HVPs),such as the cell surface markers JAG1, FRZ4, LIFR and/or FGFR3, or theintracellular marker Islet 1. Alternatively, as used herein, anengraftment marker can be a “negative marker”, which refers to thosemarkers whose expression negatively correlates with other identifyingmarkers of human ventricular progenitor cells (HVPs), such as the cellsurface markers JAG1, FRZ4, LIFR and/or FGFR3, or the intracellularmarker Islet 1.

As used herein, the term “angiogenic marker” refers to a subcategory ofengraftment marker in which the angiogenic marker is a gene or proteinknown to be involved in the process of angiogenesis.

As used herein, the term “extracellular matrix marker” refers to asubcategory of engraftment marker in which the extracellular matrixmarker is a gene or protein known to be involved in the process ofdeposition of an extracellular matrix.

As used herein, the terms “Jagged 1”, “Jag 1” and “JAG 1” are usedinterchangeably to refer to a protein known in the art that has beendescribed in, for example, Oda, T. et al. (1997) Genomics, 43:376-379;Oda, T. et al. (1997) Nat. Genet. 16:235-242; Li, L. et al. (1998)Immunity, 8:43-55; Bash, J. et al. (1999) EMBO J., 18:2803-2811; andJones, E. A. et al. (2000) J. Med. Genet. 37:658-662. A non-limitingexample of a Jagged 1 protein is the human protein having the amino acidsequence set forth in Genbank Accession Number P78504.3.

As used herein, the terms “Frizzled 4”, “Fzd 4” and “FZD 4” are usedinterchangeably to refer to a protein known in the art that has beendescribed in, for example, Kinkoshi, H. et al. (1999) Biochem. Biophys.Res. Commun., 264:955-961; Tanaka, S. et al. (1998) Proc. Natl. Acad.Sci. USA 95:10164-10169; and Robitaille, J. et al. (2002) Nat. Genet.,32:326-330. A non-limiting example of a Frizzled 4 protein is the humanprotein having the amino acid sequence set forth in Genbank AccessionNumber Q9ULV1.

As used herein, the terms “Leukemia Inhibitor Factor Receptor”, “LIFReceptor” and “LIFR” are used interchangeably to refer to a proteinknown in the art that has been described in, for example, Gearing, D. etal. (1991) EMBO J. 10:2839-2848; Gearing, D. and Bruce, A. G. (1992)New. Biol. 4:61-65; and Schiemann, W. P. et al. (1995) Proc. Natl. Acad.Sci. USA 92:5361-5365. LIFR is also referred to in the art as LeukemiaInhibitor Factor Receptor Alpha, CD118, CD118 antigen, SJ52, STWS andSWS. A non-limiting example of a LILFR protein is the human proteinhaving the amino acid sequence set forth in Genbank Accession NumberNP_001121143.1.

As used herein, the terms “Fibroblast Growth Factor Receptor 3”, “FGFReceptor 3” and “FGFR3” are used interchangeably to refer to a proteinknown in the art that has been described in, for example, Keegan, K. etal. (1991) Proc. Natl. Acad. Sci. USA 88:1095-1099; Thompson, L. M. etal. (1991) Genomics 11:1133-1142; and Shiang, R. et al. (1994) Cell78:335-343. FGFR3 is also referred to in the art as CD333, CD333antigen, EC 2.7.10.1, JTK4, ACH, CEK2 and HSFGFR3EX. A non-limitingexample of an FGFR3 protein is the human protein having the amino acidsequence set forth in Genbank Accession Number NP_000133.1.

As used herein, the term “stem cells” is used in a broad sense andincludes traditional stem cells, progenitor cells, pre-progenitor cells,reserve cells, and the like. The term “stem cell” or “progenitor” areused interchangeably herein, and refer to an undifferentiated cell whichis capable of proliferation and giving rise to more progenitor cellshaving the ability to generate a large number of mother cells that canin turn give rise to differentiated, or differentiable daughter cells.The daughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one embodiment, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types each can give rise to may vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stem-ness.” Self-renewal is the other classical part ofthe stem cell definition, and it is essential as used in this document.In theory, self-renewal can occur by either of two major mechanisms.Stem cells may divide asymmetrically, with one daughter retaining thestem state and the other daughter expressing some distinct otherspecific function and phenotype. Alternatively, some of the stem cellsin a population can divide symmetrically into two stems, thusmaintaining some stem cells in the population as a whole, while othercells in the population give rise to differentiated progeny only.Formally, it is possible that cells that begin as stem cells mightproceed toward a differentiated phenotype, but then “reverse” andre-express the stem cell phenotype, a term often referred to as“dedifferentiation”.

The term “progenitor cell” is used herein to refer to cells that have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate.

The term “pluripotent” as used herein refers to a cell with thecapacity, under different conditions, to differentiate to cell typescharacteristic of all three germ cell layers (endoderm, mesoderm andectoderm). Pluripotent cells are characterized primarily by theirability to differentiate to all three germ layers, using, for example, anude mouse and teratomas formation assay. Pluripotency is also evidencedby the expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. In someembodiments, a pluripotent cell is an undifferentiated cell.

The term “pluripotency” or a “pluripotent state” as used herein refersto a cell with the ability to differentiate into all three embryonicgerm layers: endoderm (gut tissue), mesoderm (including blood, muscle,and vessels), and ectoderm (such as skin and nerve), and typically hasthe potential to divide in vitro for a long period of time, e.g.,greater than one year or more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of multipotent cells include adult stem cells,such as for example, hematopoietic stem cells and neural stem cells.Multipotent means a stem cell may form many types of cells in a givenlineage, but not cells of other lineages. For example, a multipotentblood stem cell can form the many different types of blood cells (red,white, platelets, etc.), but it cannot form neurons.

The term “embryonic stem cell” or “ES cell” or “ESC” are usedinterchangeably herein and refer to the pluripotent stem cells of theinner cell mass of the embryonic blastocyst (see U.S. Pat. Nos.5,843,780, 6,200,806, which are incorporated herein by reference). Suchcells can similarly be obtained from the inner cell mass of blastocystsderived from somatic cell nuclear transfer (see, for example, U.S. Pat.Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein byreference). The distinguishing characteristics of an embryonic stem celldefine an embryonic stem cell phenotype. Accordingly, a cell has thephenotype of an embryonic stem cell if it possesses one or more of theunique characteristics of an embryonic stem cell such that that cell canbe distinguished from other cells. Exemplary distinguishing embryonicstem cell characteristics include, without limitation, gene expressionprofile, proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like. In someembodiments, an ES cell can be obtained without destroying the embryo,for example, without destroying a human embryo.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. Exemplary adult stem cellsinclude neural stem cells, neural crest stem cells, mesenchymal stemcells, hematopoietic stem cells, and pancreatic stem cells. As indicatedabove, stem cells have been found resident in virtually every tissue.Accordingly, the present invention appreciates that stem cellpopulations can be isolated from virtually any animal tissue.

The term “human pluripotent stem cell” (abbreviated as hPSC), as usedherein, refers to a human cell that has the capacity to differentiateinto a variety of different cell types as discussed above regarding stemcells and pluripotency. Human pluripotent human stem cells include, forexample, induced pluripotent stem cells (iPSC) and human embryonic stemcells, such as ES cell lines.

The term “human cardiac progenitor cell”, as used herein, refers to ahuman progenitor cell that is committed to the cardiac lineage and thathas the capacity to differentiate into all three cardiac lineage cells(cardiac muscle cells, endothelial cells and smooth muscle cells).

The term “human cardiomyogenic progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat predominantly differentiates into cardiac muscle cells (i.e., morethan 50% of the differentiated cells, preferably more than 60%, 70%, 80%or 90% of the differentiated cells, derived from the progenitor cellsare cardiac muscle cells).

The term “cardiac ventricular progenitor cell”, as used herein, refersto a progenitor cell that is committed to the cardiac lineage and thatpredominantly differentiates into cardiac ventricular muscle cells(i.e., more than 50% of the differentiated cells, preferably more than60%, 70%, 80% or 90% of the differentiated cells, derived from theprogenitor cells are cardiac ventricular muscle cells). This type ofcell is also referred to herein as a human ventricular progenitor, orHVP, cell.

The term “cardiomyocyte” refers to a muscle cell of the heart (e.g. acardiac muscle cell). A cardiomyocyte will generally express on its cellsurface and/or in the cytoplasm one or more cardiac-specific marker.Suitable cardiomyocyte-specific markers include, but are not limited to,cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3, GATA-4,myosin heavy chain, myosin light chain-2a, myosin light chain-2v,ryanodine receptor, and atrial natriuretic factor.

The term “derived from” used in the context of a cell derived fromanother cell means that a cell has stemmed (e.g. changed from orproduced by) a cell that is a different cell type. The term “derivedfrom” also refers to cells that have been differentiated from aprogenitor cell.

The term “Isl1+ cardiac progenitor cell”, as used herein, refers to ahuman progenitor cell that is committed to the cardiac lineage and thatexpresses Islet 1.

The term “Isl1+ JAG1+ cardiac progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat expresses both Islet 1 and Jagged 1.

The term “Isl1+ FZD4+ cardiac progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat expresses both Islet 1 and Frazzled 4.

The term “Isl1+ LIFR+ cardiac progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat expresses both Islet 1 and LIFR.

The term “Isl1+ FGFR3+ cardiac progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat expresses both Islet 1 and FGFR3.

The term “Isl1+ TNFSF9+ cardiac progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat expresses both Islet 1 and TNFSF9.

The term “Isl1+ JAG1+ FZD4+ LIFR+ FGFR3+ TNFSF9+ cardiac progenitorcell”, as used herein, refers to a human progenitor cell that iscommitted to the cardiac lineage and that expresses Islet 1, JAG1, FZD4,LIFR, FGFR3 and TNFSF9.

With respect to cells in cell cultures or in cell populations, the term“substantially free of” means that the specified cell type of which thecell culture or cell population is free, is present in an amount of lessthan about 10%, less than about 9%, less than about 8%, less than about7%, less than about 6%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2% or less than about 1% of the totalnumber of cells present in the cell culture or cell population.

In the context of cell ontogeny, the adjective “differentiated”, or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as an cardiomyocyte precursor), and thento an end-stage differentiated cell, which plays a characteristic rolein a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “differentiation” in the present context means the formation ofcells expressing markers known to be associated with cells that are morespecialized and closer to becoming terminally differentiated cellsincapable of further differentiation. The pathway along which cellsprogress from a less committed cell, to a cell that is increasinglycommitted to a particular cell type, and eventually to a terminallydifferentiated cell is referred to as progressive differentiation orprogressive commitment. Cell which are more specialized (e.g., havebegun to progress along a path of progressive differentiation) but notyet terminally differentiated are referred to as partiallydifferentiated. Differentiation is a developmental process whereby cellsassume a specialized phenotype, e.g., acquire one or morecharacteristics or functions distinct from other cell types. In somecases, the differentiated phenotype refers to a cell phenotype that isat the mature endpoint in some developmental pathway (a so calledterminally differentiated cell). In many, but not all tissues, theprocess of differentiation is coupled with exit from the cell cycle. Inthese cases, the terminally differentiated cells lose or greatlyrestrict their capacity to proliferate. However, we note that in thecontext of this specification, the terms “differentiation” or“differentiated” refer to cells that are more specialized in their fateor function than at a previous point in their development, and includesboth cells that are terminally differentiated and cells that, althoughnot terminally differentiated, are more specialized than at a previouspoint in their development. The development of a cell from anuncommitted cell (for example, a stem cell), to a cell with anincreasing degree of commitment to a particular differentiated celltype, and finally to a terminally differentiated cell is known asprogressive differentiation or progressive commitment. A cell that is“differentiated” relative to a progenitor cell has one or morephenotypic differences relative to that progenitor cell. Phenotypicdifferences include, but are not limited to morphologic differences anddifferences in gene expression and biological activity, including notonly the presence or absence of an expressed marker, but alsodifferences in the amount of a marker and differences in theco-expression patterns of a set of markers.

The term “differentiation” as used herein refers to the cellulardevelopment of a cell from a primitive stage towards a more mature (i.e.less primitive) cell.

As used herein, “proliferating” and “proliferation” refers to anincrease in the number of cells in a population (growth) by means ofcell division. Cell proliferation is generally understood to result fromthe coordinated activation of multiple signal transduction pathways inresponse to the environment, including growth factors and othermitogens. Cell proliferation may also be promoted by release from theactions of intra- or extracellular signals and mechanisms that block ornegatively affect cell proliferation.

The terms “renewal” or “self-renewal” or “proliferation” are usedinterchangeably herein, and refers to a process of a cell making morecopies of itself (e.g. duplication) of the cell. In some embodiments,cells are capable of renewal of themselves by dividing into the sameundifferentiated cells (e.g. progenitor cell type) over long periods,and/or many months to years. In some instances, proliferation refers tothe expansion of cells by the repeated division of single cells into twoidentical daughter cells.

The term “lineages” as used herein refers to a term to describe cellswith a common ancestry or cells with a common developmental fate, forexample cells that have a developmental fate to develop into ventricularcardiomyocytes.

The term “clonal population”, as used herein, refers to a population ofcells that is derived from the outgrowth of a single cell. That is, thecells within the clonal population are all progeny of a single cell thatwas used to seed the clonal population.

The term “media” as referred to herein is a medium for maintaining atissue or cell population, or culturing a cell population (e.g. “culturemedia”) containing nutrients that maintain cell viability and supportproliferation. The cell culture medium may contain any of the followingin an appropriate combination: salt(s), buffer(s), amino acids, glucoseor other sugar(s), antibiotics, serum or serum replacement, and othercomponents such as peptide growth factors, etc. Cell culture mediaordinarily used for particular cell types are known to those skilled inthe art.

The term “phenotype” refers to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

A “marker” as used herein describes the characteristics and/or phenotypeof a cell. Markers can be used for selection of cells comprisingcharacteristics of interest. Markers will vary with specific cells.Markers are characteristics, whether morphological, functional orbiochemical (enzymatic) characteristics particular to a cell type, ormolecules expressed by the cell type. Such markers can be proteins, forexample a cell surface protein that possesses an epitope for antibodiesor other binding molecules available in the art. A marker may consist ofany molecule found in a cell including, but not limited to, DNA, RNA,mRNA, proteins (peptides and polypeptides), lipids, polysaccharides,nucleic acids and steroids. Examples of morphological characteristics ortraits include, but are not limited to, shape, size, and nuclear tocytoplasmic ratio. Examples of functional characteristics or traitsinclude, but are not limited to, the ability to adhere to particularsubstrates, ability to incorporate or exclude particular dyes, abilityto migrate under particular conditions, and the ability to differentiatealong particular lineages. Markers may be detected by any methodavailable to one of skill in the art.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a substantially purepopulation of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched from.

The term “substantially pure”, with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, to whom cardiac ventricularprogenitor cells as disclosed herein can be implanted into, for e.g.treatment, which in some embodiments encompasses prophylactic treatmentor for a disease model, with methods and compositions described herein,is or are provided. For treatment of disease states that are specificfor a specific animal such as a human subject, the term “subject” refersto that specific animal. The terms “non-human animals” and “non-humanmammals” are used interchangeably herein, and include mammals such asrats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates. The term “subject” also encompasses any vertebrate includingbut not limited to mammals, reptiles, amphibians and fish. However,advantageously, the subject is a mammal such as a human, or othermammals such as a domesticated mammal, e.g. dog, cat, horse, and thelike, or production mammal, e.g. cow, sheep, pig, and the like are alsoencompassed in the term subject.

As used herein, the term “recipient” refers to a subject that willreceive a transplanted organ, tissue or cell.

The term “three-dimensional matrix” or “scaffold” or “matrices” as usedherein refers in the broad sense to a composition comprising abiocompatible matrix, scaffold, or the like. The three-dimensionalmatrix may be liquid, gel, semi-solid, or solid at 25° C. Thethree-dimensional matrix may be biodegradable or non-biodegradable. Insome embodiments, the three-dimensional matrix is biocompatible, orbioresorbable or bioreplacable. Exemplary three-dimensional matricesinclude polymers and hydrogels comprising collagen, fibrin, chitosan,MATRIGEL™, polyethylene glycol, dextrans including chemicallycrosslinkable or photocrosslinkable dextrans, processed tissue matrixsuch as submucosal tissue and the like. In certain embodiments, thethree-dimensional matrix comprises allogeneic components, autologouscomponents, or both allogeneic components and autologous components. Incertain embodiments, the three-dimensional matrix comprises synthetic orsemi-synthetic materials. In certain embodiments, the three-dimensionalmatrix comprises a framework or support, such as a fibrin-derivedscaffold.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement ofcardiomyogenic progenitor cells and/or cardiomyocytes differentiated asdescribed herein into a subject by a method or route which results in atleast partial localization of the cells at a desired site. The cells canbe administered by any appropriate route that results in delivery to adesired location in the subject where at least a portion of the cellsremain viable. The period of viability of the cells after administrationto a subject can be as short as a few hours, e.g. twenty-four hours, toa few days, to as long as several years.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value. The term “substantially” or “predominantly” as usedherein means a proportion of at least about 60%, or preferably at leastabout 70% or at least about 80%, or at least about 90%, at least about95%, at least about 97% or at least about 99% or more, or any integerbetween 70% and 100%.

The term “disease” or “disorder” is used interchangeably herein, andrefers to any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, indisposition oraffection.

As used herein, the phrase “cardiovascular condition, disease ordisorder” is intended to include all disorders characterized byinsufficient, undesired or abnormal cardiac function, e.g. ischemicheart disease, hypertensive heart disease and pulmonary hypertensiveheart disease, valvular disease, congenital heart disease and anycondition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death. As used herein, the term “ischemia”refers to any localized tissue ischemia due to reduction of the inflowof blood. The term “myocardial ischemia” refers to circulatorydisturbances caused by coronary atherosclerosis and/or inadequate oxygensupply to the myocardium. For example, an acute myocardial infarctionrepresents an irreversible ischemic insult to myocardial tissue. Thisinsult results in an occlusive (e.g., thrombotic or embolic) event inthe coronary circulation and produces an environment in which themyocardial metabolic demands exceed the supply of oxygen to themyocardial tissue.

As used herein, the term “treating” or “treatment” are usedinterchangeably herein and refers to reducing or decreasing oralleviating or halting at least one adverse effect or symptom of acardiovascular condition, disease or disorder, i.e., any disordercharacterized by insufficient or undesired cardiac function. Adverseeffects or symptoms of cardiac disorders are well-known in the art andinclude, but are not limited to, dyspnea, chest pain, palpitations,dizziness, syncope, edema, cyanosis, pallor, fatigue and death. In someembodiments, the term “treatment” as used herein refers to prophylactictreatment or preventative treatment to prevent the development of asymptom of a cardiovascular condition in a subject.

Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced as that term is defined herein. Alternatively, atreatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or decrease of markers of the disease, but also a cessation orslowing of progress or worsening of a symptom that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already diagnosedwith a cardiac condition, as well as those likely to develop a cardiaccondition due to genetic susceptibility or other factors such as weight,diet and health. In some embodiments, the term to treat also encompassespreventative measures and/or prophylactic treatment, which includesadministering a pharmaceutical composition as disclosed herein toprevent the onset of a disease or disorder.

A therapeutically significant reduction in a symptom is, e.g. at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, at least about 125%,at least about 150% or more in a measured parameter as compared to acontrol or non-treated subject. Measured or measurable parametersinclude clinically detectable markers of disease, for example, elevatedor depressed levels of a biological marker, as well as parametersrelated to a clinically accepted scale of symptoms or markers for adisease or disorder. It will be understood, that the total daily usageof the compositions and formulations as disclosed herein will be decidedby the attending physician within the scope of sound medical judgment.The exact amount required will vary depending on factors such as thetype of disease being treated.

With reference to the treatment of a cardiovascular condition or diseasein a subject, the term “therapeutically effective amount” refers to theamount that is safe and sufficient to prevent or delay the developmentor a cardiovascular disease or disorder. The amount can thus cure orcause the cardiovascular disease or disorder to go into remission, slowthe course of cardiovascular disease progression, slow or inhibit asymptom of a cardiovascular disease or disorder, slow or inhibit theestablishment of secondary symptoms of a cardiovascular disease ordisorder or inhibit the development of a secondary symptom of acardiovascular disease or disorder. The effective amount for thetreatment of the cardiovascular disease or disorder depends on the typeof cardiovascular disease to be treated, the severity of the symptoms,the subject being treated, the age and general condition of the subject,the mode of administration and so forth. Thus, it is not possible tospecify the exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation. The efficacy oftreatment can be judged by an ordinarily skilled practitioner, forexample, efficacy can be assessed in animal models of a cardiovasculardisease or disorder as discussed herein, for example treatment of arodent with acute myocardial infarction or ischemia-reperfusion injury,and any treatment or administration of the compositions or formulationsthat leads to a decrease of at least one symptom of the cardiovasculardisease or disorder as disclosed herein, for example, increased heartejection fraction, decreased rate of heart failure, decreased infarctsize, decreased associated morbidity (pulmonary edema, renal failure,arrhythmias) improved exercise tolerance or other quality of lifemeasures, and decreased mortality indicates effective treatment. Inembodiments where the compositions are used for the treatment of acardiovascular disease or disorder, the efficacy of the composition canbe judged using an experimental animal model of cardiovascular disease,e.g., animal models of ischemia-reperfusion injury (Headrick J P, Am JPhysiol Heart circ Physiol 285; H1797; 2003) and animal models acutemyocardial infarction. (Yang Z, Am J Physiol Heart Circ. Physiol282:H949:2002; Guo Y, J Mol Cell Cardiol 33; 825-830, 2001). When usingan experimental animal model, efficacy of treatment is evidenced when areduction in a symptom of the cardiovascular disease or disorder, forexample, a reduction in one or more symptom of dyspnea, chest pain,palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue andhigh blood pressure which occurs earlier in treated, versus untreatedanimals. By “earlier” is meant that a decrease, for example in the sizeof the tumor occurs at least 5% earlier, but preferably more, e.g., oneday earlier, two days earlier, 3 days earlier, or more.

As used herein, the term “treating” when used in reference to atreatment of a cardiovascular disease or disorder is used to refer tothe reduction of a symptom and/or a biochemical marker of acardiovascular disease or disorder, for example a reduction in at leastone biochemical marker of a cardiovascular disease by at least about 10%would be considered an effective treatment. Examples of such biochemicalmarkers of cardiovascular disease include a reduction of, for example,creatine phosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) in the blood, and/or a decrease in a symptom ofcardiovascular disease and/or an improvement in blood flow and cardiacfunction as determined by someone of ordinary skill in the art asmeasured by electrocardiogram (ECG or EKG), or echocardiogram (heartultrasound), Doppler ultrasound and nuclear medicine imaging. Areduction in a symptom of a cardiovascular disease by at least about 10%would also be considered effective treatment by the methods as disclosedherein. As alternative examples, a reduction in a symptom ofcardiovascular disease, for example a reduction of at least one of thefollowing; dyspnea, chest pain, palpitations, dizziness, syncope, edema,cyanosis etc. by at least about 10% or a cessation of such systems, or areduction in the size one such symptom of a cardiovascular disease by atleast about 10% would also be considered as affective treatments by themethods as disclosed herein. In some embodiments, it is preferred, butnot required that the therapeutic agent actually eliminate thecardiovascular disease or disorder, rather just reduce a symptom to amanageable extent.

Subjects amenable to treatment by the methods as disclosed herein can beidentified by any method to diagnose myocardial infarction (commonlyreferred to as a heart attack) commonly known by persons of ordinaryskill in the art are amenable to treatment using the methods asdisclosed herein, and such diagnostic methods include, for example butare not limited to; (i) blood tests to detect levels of creatinephosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) and other enzymes released during myocardialinfarction; (ii) electrocardiogram (ECG or EKG) which is a graphicrecordation of cardiac activity, either on paper or a computer monitor.An ECG can be beneficial in detecting disease and/or damage; (iii)echocardiogram (heart ultrasound) used to investigate congenital heartdisease and assessing abnormalities of the heart wall, includingfunctional abnormalities of the heart wall, valves and blood vessels;(iv) Doppler ultrasound can be used to measure blood flow across a heartvalve; (v) nuclear medicine imaging (also referred to as radionuclidescanning in the art) allows visualization of the anatomy and function ofan organ, and can be used to detect coronary artery disease, myocardialinfarction, valve disease, heart transplant rejection, check theeffectiveness of bypass surgery, or to select patients for angioplastyor coronary bypass graft.

The terms “coronary artery disease” and “acute coronary syndrome” asused interchangeably herein, and refer to myocardial infarction refer toa cardiovascular condition, disease or disorder, include all disorderscharacterized by insufficient, undesired or abnormal cardiac function,e.g. ischemic heart disease, hypertensive heart disease and pulmonaryhypertensive heart disease, valvular disease, congenital heart diseaseand any condition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death.

As used herein, the term “ischemia” refers to any localized tissueischemia due to reduction of the inflow of blood. The term “myocardialischemia” refers to circulatory disturbances caused by coronaryatherosclerosis and/or inadequate oxygen supply to the myocardium. Forexample, an acute myocardial infarction represents an irreversibleischemic insult to myocardial tissue. This insult results in anocclusive (e.g., thrombotic or embolic) event in the coronarycirculation and produces an environment in which the myocardialmetabolic demands exceed the supply of oxygen to the myocardial tissue.

The terms “composition” or “pharmaceutical composition” usedinterchangeably herein refer to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. In someembodiments, pharmaceutical compositions can be specifically formulatedfor direct delivery to a target tissue or organ, for example, by directinjection or via catheter injection to a target tissue. In otherembodiments, compositions can be specifically formulated foradministration via one or more of a number of routes, including but notlimited to, oral, ocular parenteral, intravenous, intraarterial,subcutaneous, intranasal, sublingual, intraspinal,intracerebroventricular, and the like. In addition, compositions fortopical (e.g., oral mucosa, respiratory mucosa) and/or oraladministration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement of apharmaceutical composition comprising cardiomyogenic progenitor cells,or a composition comprising a population of differentiatedcardiomyocytes (e.g., ventricular cardiomyocytes) as described herein,into a subject by a method or route which results in at least partiallocalization of the pharmaceutical composition, at a desired site ortissue location. In some embodiments, the pharmaceutical composition canbe administered by any appropriate route which results in effectivetreatment in the subject, i.e. administration results in delivery to adesired location or tissue in the subject where at least a portion ofthe cells are located at a desired target tissue or target celllocation.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of cardiovascular stem cells and/or their progeny and/orcompound and/or other material other than directly into the cardiactissue, such that it enters the animal's system and, thus, is subject tometabolism and other like processes, for example, subcutaneous orintravenous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation.

The term “drug” or “compound” or “test compound” as used herein refersto a chemical entity or biological product, or combination of chemicalentities or biological products, administered to a subject to treat orprevent or control a disease or condition. The chemical entity orbiological product is preferably, but not necessarily a low molecularweight compound, but may also be a larger compound, for example, anoligomer of nucleic acids, amino acids, or carbohydrates includingwithout limitation proteins, oligonucleotides, ribozymes, DNAzymes,glycoproteins, siRNAs, lipoproteins, aptamers, and modifications andcombinations thereof.

The term “transplantation” as used herein refers to introduction of newcells (e.g. reprogrammed cells), tissues (such as differentiated cellsproduced from reprogrammed cells), or organs into a host (i.e.transplant recipient or transplant subject)

The terms “agent reactive with JAG1”, “agent reactive with FZD4”, “agentreactive with LIFR”, “agent that reacts with FGFR3” and “agent reactivewith TNFSF9”, as used herein, refers to an agent that binds to orotherwise interacts with JAG1, FZD4, LIFR, FGFR3 or TNFSF9,respectively. Preferably, the agent “specifically” binds or otherwiseinteracts with JAG1, FZD4, LIFR, FGFR3 or TNFSF9, respectively, suchthat it does not bind or interact with other proteins.

The term “agent reactive with Islet 1”, as used herein, refers to anagent that binds to or otherwise interacts with Islet 1. Preferably, theagent “specifically” binds or otherwise interacts with Islet 1 such thatit does not bind or interact with other non-Islet 1 proteins.

The term “antibody”, as used herein, includes whole antibodies and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in one preferred embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The term“antigen-binding portion” of an antibody (or simply “antibody portion”),as used herein, refers to one or more fragments of an antibody thatretain the ability to specifically bind to an antigen.

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope.

The term “human monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which hasvariable and optional constant regions derived from human germlineimmunoglobulin sequences.

The term “humanized monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain CDR1, 2 and 3 from a non-human antibody (e.g., a mousemonoclonal antibody) grafted into human framework and constant regions.

The term “chimeric monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain variable regions from one species linked to constantregions from another species.

The term “fusion protein”, as used herein, refers to a compositeprotein, typically made using recombinant DNA technology, in which twodifferent proteins, or portions thereof, are operatively linkedtogether. A non-limiting example is an Fc fusion protein in which anon-immunoglobulin protein is operatively linked to an immunoglobulin Fcregion.

Various aspects of the invention are described in further detail in thefollowing subsections.

Methods of Identifying and Transplanting Engraftable Human VentricularProgenitor Cells

It has been determined that engraftable HVPs typically develop betweendays 5-7 of differentiation (e.g., day 6) under the culture conditionsdescribed below and in Examples 1 and 11, express the intracellularmarker Islet 1 and express cell surface markers including JAG1, FZD4,LIFR, FGFR3 and/or TNFSF9 (see e.g., U.S. Ser. No. 14/832,324, filedAug. 21, 2015, and U.S. Ser. No. 14/984,783, filed Dec. 30, 2015, theentire contents of each of which are expressly incorporated herein byreference). To further characterize the gene expression profile ofengraftable HVPs, RNA sequencing was performed at different time pointson HVPs undergoing differentiation, as described in Examples 12 and 13.Cluster analysis of gene expression profiles at different time pointswas used to identify stage-specific signature genes. Gene ontogenysearches then allowed for the identification of genes (angiogenic familygenes and extracellular matrix family genes) critical for cellengraftment, referred to herein as engraftment markers.

In one embodiment, the engraftment marker is an angiogenic marker. In aparticular embodiment, the angiogenic marker is a positive angiogenicmarker. Table 1 lists those angiogenic genes whose expression profilestrongly correlates with Islet 1 (Isl1) expression in the HVPs (aPearson correlation with Isl1 expression of 0.50 or greater). Table 2lists additional angiogenic genes whose expression profile correlateswith Isl1 expression in HVPs (a Pearson correlation with Isl1 expressionbetween 0.49 and 0.00). In another particular embodiment, the angiogenicmarker is a negative angiogenic marker. Table 3 lists those angiogenicgenes whose expression profile strongly negatively correlates with Islet1 (Isl1) expression in the HVPs (a Pearson correlation with Isl1expression of −0.50 or less). Table 4 lists additional angiogenic geneswhose expression profile negatively correlates with Isl1 expression inHVPs (a Pearson correlation with Isl1 expression between 0.00 and−0.49).

In one embodiment, the engraftment marker is an extracellular matrixmarker. In a particular embodiment, the extracellular marker is apositive extracellular matrix marker. Table 5 lists those extracellularmatrix genes whose expression profile strongly correlates with Islet 1(Isl1) expression in the HVPs (a Pearson correlation with Isl1expression of 0.50 or greater). Table 6 lists additional extracellularmatrix genes whose expression profile correlates with Isl1 expression inHVPs (a Pearson correlation with Isl1 expression between 0.49 and 0.00).In another particular embodiment, the extracellular matrix marker is anegative extracellular matrix marker. Table 7 lists those extracellularmatrix genes whose expression profile strongly negatively correlateswith Islet 1 (Isl1) expression in the HVPs (a Pearson correlation withIsl1 expression of −0.50 or less). Table 8 lists additionalextracellular matrix genes whose expression profile negativelycorrelates with Isl1 expression in HVPs (a Pearson correlation with Isl1expression between 0.00 and −0.49).

Accordingly, in one aspect, the invention provides a method ofidentifying engraftable human ventricular progenitor cells (HVPs), themethod comprising:

-   -   detecting expression of at least one engraftment marker in the        HVPs to thereby identify engraftable HVPs, wherein:    -   the HVPs also express at least one surface marker selected from        the group consisting of: JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.

In another embodiment, the invention provides a method of isolatingengraftable human ventricular progenitor cells (HVPs), the methodcomprising:

-   -   culturing human cells containing cardiac progenitor cells under        conditions causing differentiation into human ventricular        progenitor cells (HVPs);    -   detecting expression on the HVPs of at least one surface marker        selected from the group consisting of JAG1, FZD4, LIFR, FGFR3        and/or TNFSF9;    -   detecting expression in the HVPs of at least one engraftment        marker; and    -   isolating HVPs that co-express the at least one surface marker        and the at least one engraftment marker to thereby isolate        engraftable HVPs.

In another embodiment, the invention provides a method for engraftinghuman ventricular tissue in a subject, the method comprising:

-   -   transplanting engraftable human ventricular progenitor cells        (HVPs) into the subject such that human ventricular tissue is        engrafted in the subject,    -   wherein prior to transplantation, engraftable HVPs are        identified by detecting expression of at least one engraftment        marker in the HVPs; and    -   wherein the HVPs express at least one surface marker selected        from the group consisting of: JAG1, FZD4, LIFR, FGFR3 and/or        TNFSF9.

In another embodiment, the invention provides a method for engraftinghuman ventricular tissue in a subject, the method comprising:

-   -   culturing human cells containing cardiac progenitor cells under        conditions causing differentiation into human ventricular        progenitor cells (HVPs);    -   detecting expression on the HVPs of at least one surface marker        selected from the group consisting of JAG1, FZD4, LIFR, FGFR3        and/or TNFSF9;    -   detecting expression in the HVPs of at least one engraftment        marker;    -   isolating HVPs that co-express the at least one surface marker        and the at least one engraftment marker to thereby isolate        engraftable HVPs; and    -   transplanting engraftable the HVPs into the subject such that        human ventricular tissue is engrafted in the subject.

In another embodiment, the invention provides a method for engraftinghuman ventricular tissue in a subject, the method comprising:

-   -   culturing human cells containing cardiac progenitor cells under        conditions causing differentiation into human ventricular        progenitor cells (HVPs) expressing at least one surface marker        selected from the group consisting of JAG1, FZD4, LIFR, FGFR3        and/or TNFSF9;    -   isolating the HVPs expressing the at least one surface marker to        form an HVP population;    -   detecting expression of at least one engraftment marker in a        sample of the HVP population; and    -   transplanting the HVPs expressing the at least one surface        marker and the at least one engraftment marker into the subject        such that human ventricular tissue is engrafted in the subject.

In another embodiment, the invention provides a method for engraftinghuman ventricular tissue in a subject, the method comprising:

-   -   contacting a culture of human cells containing human ventricular        progenitor cells (HVPs) with at least one agent reactive with at        least one surface marker selected from the group consisting of        JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9 such that a population of        HVPs is isolated;    -   expanding a clonal population of HVPs from the isolated        population of HVPs;    -   detecting expression of at least one engraftment marker in a        sample of the clonal population of HVPs; and    -   transplanting the clonal population of HVPs expressing the at        least one surface marker and the at least one engraftment marker        into the subject such that human ventricular tissue is engrafted        in the subject.

In one embodiment, the at least one engraftment marker detected is atleast one positive angiogenic marker. More than one positive angiogenicmarker can be detected, for example, two or more, three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, ten or more, fifteen or more, or twenty or more positiveangiogenic markers can be detected.

In another embodiment, the at least one engraftment marker detected isat least one positive extracellular matrix marker. More than onepositive extracellular marker can be detected, for example, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, fifteen or more, or twenty ormore positive extracellular markers can be detected.

In another embodiment, the at least one engraftment marker detected isat least one positive angiogenic marker and at least one positiveextracellular matrix marker. More than one of each positive marker(angiogenic and extracellular matrix) can be detected, for example, twoor more, three or more, four or more, five or more, six or more, sevenor more, eight or more, nine or more, ten or more, fifteen or more, ortwenty or more positive angiogenic and extracellular markers each can bedetected.

In one embodiment, the at least one positive angiogenic marker isselected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA,EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1,ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT,NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1,HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1,NRXN3, FZD5 and HHEX. In another embodiment, the at least one positiveangiogenic marker is selected from the group consisting of: FGF10,PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1,MEIS1, TBX1, PKNOX1, ID1, TCF21 and HEY1. In another embodiment, the atleast positive angiogenic marker is selected from the group consistingof: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2 and NTRK1. In anotherembodiment, the positive angiogenic marker is FGF10. In anotherembodiment, the positive angiogenic marker is PRKD1. In anotherembodiment, the positive angiogenic marker is CCBE1. In anotherembodiment, the positive angiogenic marker is PDGFRA. In anotherembodiment, the positive angiogenic marker is EPHB2. In anotherembodiment, the positive angiogenic marker is GATA2. In anotherembodiment, the positive angiogenic marker is NTRK1. In anotherembodiment, the positive angiogenic marker is PTGIS. In anotherembodiment, the positive angiogenic marker is BMPER. In anotherembodiment, the positive angiogenic marker is BMP4.

In another embodiment, the at least one positive extracellular matrixmarker is selected from the group consisting of: FGF10, SMOC1, CCBE1,COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1,HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD,MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4,CRTAC1, LTBP4 and FREM1. In another embodiment, the at least onepositive extracellular matrix marker is selected from the groupconsisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1,BMP4, FBLN7, FBLN2, NDNF and HTRA1. In another embodiment, the at leastone positive extracellular matrix marker is selected from the groupconsisting of: FGF10, SMOC1 and CCBE1. In another embodiment, thepositive extracellular marker is FGF10. In another embodiment, thepositive extracellular marker is SMOC1. In another embodiment, thepositive extracellular marker is CCBE1. In another embodiment, thepositive extracellular marker is COL6A6. In another embodiment, thepositive extracellular marker is ADAMTS12. In another embodiment, thepositive extracellular marker is COL19A1. In another embodiment, thepositive extracellular marker is LAMA1. In another embodiment, thepositive extracellular marker is BMP4. In another embodiment, thepositive extracellular marker is FBLN7. In another embodiment, thepositive extracellular marker is FBLN2.

In those embodiments in which both at least one positive angiogenicmarker and at least one positive extracellular matrix marker aredetected, the at least one positive angiogenic marker can be selectedfrom the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2,NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21,HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN,SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2,CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5and HHEX; and

the at least one positive extracellular matrix marker can be selectedfrom the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12,COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1,SPOCK3, PODNL1, IHH, ALAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1,HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 andFREM1.

In another embodiment, the at least one positive angiogenic marker isselected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA,EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1,ID1, TCF21 and HEY1; and

the at least one positive extracellular matrix marker is selected fromthe group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1,LAMA1, BMP4, FBLN7, FBLN2, NDNF and HTRA1.

In another embodiment, the at least one positive angiogenic marker isselected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA,EPHB2, GATA2 and NTRK1; and

the at least one positive extracellular matrix marker is selected fromthe group consisting of: FGF10, SMOC1 and CCBE1.

In one embodiment, the at least one engraftment marker detected is atleast one negative angiogenic marker. More than one negative angiogenicmarker can be detected, for example, two or more, three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, ten or more, fifteen or more, or twenty or more negativeangiogenic markers can be detected.

In another embodiment, the at least one engraftment marker detected isat least one negative extracellular matrix marker. More than onenegative extracellular matrix marker can be detected, for example, twoor more, three or more, four or more, five or more, six or more, sevenor more, eight or more, nine or more, ten or more, fifteen or more, ortwenty or more negative extracellular matrix markers can be detected.

In another embodiment, the at least one engraftment marker detected isat least one negative angiogenic marker and at least one negativeextracellular matrix marker. More than one of each negative marker(angiogenic and extracellular matrix) can be detected, for example, twoor more, three or more, four or more, five or more, six or more, sevenor more, eight or more, nine or more, ten or more, fifteen or more, ortwenty or more negative angiogenic and extracellular matrix markers eachcan be detected.

In one embodiment, the at least one negative angiogenic marker isselected from the group consisting of: ETS1, BAX, XBP1, TDGF1, C5AR1,EPHA1, HS6ST1, SHC1, SP100, JAM3, CASP8, FLT4, SFRP2, HPSE, BAK1, GPX1,VAV3, VAV2, EGF, ADAM15 and AGGF1. In another embodiment, the at leastone negative angiogenic marker is selected from the group consisting of:VAV3, VAV2, EGF, ADAM15 and AGGF1.

In another embodiment, the at least one negative extracellular matrixmarker is selected from the group consisting of: FKBP1A, CLU, TFP12,PLSCR1, FBLN5, VWA1, ADAMTS16, MMP25, SFRP2 and SOD1.

In those embodiments in which both at least one negative angiogenicmarker and at least one negative extracellular matrix marker aredetected, the at least one negative angiogenic marker can be selectedfrom the group consisting of: ETS1, BAX, XBP1, TDGF1, C5AR1, EPHA1,HS6ST1, SHC1, SP100, JAM3, CASP8, FLT4, SFRP2, HPSE, BAK1, GPX1, VAV3,VAV2, EGF, ADAM15 and AGGF1; and

the at least one negative extracellular matrix marker can be selectedfrom the group consisting of: FKBP1A, CLU, TFP12, PLSCR1, FBLN5, VWA1,ADAMTS16, MMP25, SFRP2 and SOD1.

The ordinarily skilled artisan will appreciate that the engraftmentmarkers of the invention can be detected by any suitable method ofdetection known in the art for detecting nucleic acid or proteinexpression. Typically, mRNA encoding the engraftment marker(s) isdetected, either directly or indirectly (e.g., by detection of cDNAprepared from the mRNA). Methods for detecting mRNAs or cDNAs are wellestablished in the art, including but not limited to hybridizationtechniques, polymerase chain reaction, quantitative PCR, real-timequantitative PCR, RNA sequencing and the like. Methods for detectingprotein expression also are well established in the art, including butnot limited to western blotting, immunoprecipitation,immunohistochemistry, ELISA and the like. Accordingly, in oneembodiment, to “detect” expression of an engraftment marker(s) in apopulation of HVPs, a sample of HVPs is taken from the population ofHVPs, nucleic acid (e.g., mRNA, cDNA) is prepared from the sample andcontacted with, for example, one or more pairs of oligonucleotideprimers capable of amplifying nucleic acid encoding the engraftmentmarker(s) of interest, following by carrying out a PCR-type reaction(e.g., standard PCR, quantitative PCR, real-time Q-PCR) on the nucleicacid sample. Alternatively, the nucleic acid sample prepared from thepopulation of HVPs can be contacted with one or more hybridizationprobes capable of binding to nucleic acid encoding the engraftmentmarker(s) of interest, followed by carrying out a hybridization reactionon the sample (e.g., northern blotting, southern blotting etc.). Inanother embodiment, to “detect” expression of an engraftment marker(s)in a population of HVPs, a sample of HVPs is taken from the populationof HVPs, protein is prepared from the sample and contacted with, forexample, one or more monoclonal antibodies that specifically bind to theengraftment marker(s) of interest, followed by carrying out animmunodetection method (e.g., western blotting, immunoprecipitation,ELISA etc.).

Suitable methods for determining the level of engraftment markerexpression at the mRNA level include RNA sequencing, conventionalmicroarray analysis, polymerase chain reaction (PCR), quantitativepolymerase chain reaction (Q-PCR) and real-time quantitative PCR(RT-QPCR). In some embodiments, RNA is extracted from the HVPs usingstandard protocols. In other embodiments, RNA analysis is performedusing techniques that do not require RNA isolation.

Methods for rapid and efficient extraction of eukaryotic mRNA, i.e.,poly(a) RNA, from tissue samples are well established and known to thoseof skill in the art. See, e.g., Ausubel et al., 1997, Current Protocolsof Molecular Biology, John Wiley & Sons. The use of commerciallyavailable kits with vendor's instructions for RNA extraction andpreparation is widespread and common. Commercial vendors of various RNAisolation products and complete kits include Qiagen (Valencia, Calif.),Invitrogen (Carlsbad, Calif.), Ambion (Austin, Tex.) and Exiqon (Woburn,Mass.).

In general, RNA isolation begins with cell disruption. During celldisruption it is desirable to minimize RNA degradation by RNases. Oneapproach to limiting RNase activity during the RNA isolation process isto ensure that a denaturant is in contact with cellular contents as soonas the cells are disrupted. Another common practice is to include one ormore proteases in the RNA isolation process. Optionally, fresh tissuesamples are immersed in an RNA stabilization solution, at roomtemperature, as soon as they are collected. The stabilization solutionrapidly permeates the cells, stabilizing the RNA for storage at 4° C.,for subsequent isolation. One such stabilization solution is availablecommercially as RNAlater® (Ambion, Austin, Tex.).

In some protocols, total RNA is isolated from disrupted cells by cesiumchloride density gradient centrifugation. In general, mRNA makes upapproximately 1% to 5% of total cellular RNA. Immobilized Oligo(dT),e.g., oligo(dT) cellulose, is commonly used to separate mRNA fromribosomal RNA and transfer RNA. If stored after isolation, RNA must bestored under RNase-free conditions. Methods for stable storage ofisolated RNA are known in the art. Various commercial products forstable storage of RNA are available.

The mRNA expression level of engraftment markers can be measured usingconventional DNA microarray expression profiling technology. A DNAmicroarray is a collection of specific DNA segments or probes affixed toa solid surface or substrate such as glass, plastic or silicon, witheach specific DNA segment occupying a known location in the array.Hybridization with a sample of labeled RNA, usually under stringenthybridization conditions, allows detection and quantitation of RNAmolecules corresponding to each probe in the array. After stringentwashing to remove non-specifically bound sample material, the microarrayis scanned by confocal laser microscopy or other suitable detectionmethod. Modern commercial DNA microarrays, often known as DNA chips,typically contain tens of thousands of probes, and thus can measureexpression of tens of thousands of genes simultaneously. Suchmicroarrays can be used in practicing the present invention.Alternatively, custom chips containing as few probes as those needed tomeasure engraftment markers of interest, plus necessary controls orstandards, e.g., for data normalization, can be used in practicing thedisclosed methods.

To facilitate data normalization, a two-color microarray reader can beused. In a two-color (two-channel) system, samples are labeled with afirst fluorophore that emits at a first wavelength, while an RNA or cDNAstandard is labeled with a second fluorophore that emits at a differentwavelength. For example, Cy3 (570 nm) and Cy5 (670 nm) often areemployed together in two-color microarray systems.

DNA microarray technology is well-developed, commercially available, andwidely employed. Therefore, in performing disclosed methods, a person ofordinary skill in the art can use microarray technology to measureexpression levels of genes encoding engraftment markers without undueexperimentation. DNA microarray chips, reagents (such as those for RNAor cDNA preparation, RNA or cDNA labeling, hybridization and washingsolutions), instruments (such as microarray readers) and protocols arewell known in the art and available from various commercial sources.Commercial vendors of microarray systems include Agilent Technologies(Santa Clara, Calif.) and Affymetrix (Santa Clara, Calif.), but otherPCR systems can be used.

The level of mRNA encoding an engraftment marker(s) can be measuredusing conventional quantitative reverse transcriptase polymerase chainreaction (qRT-PCR) technology. Advantages of qRT-PCR includesensitivity, flexibility, quantitative accuracy, and ability todiscriminate between closely related mRNAs. Guidance concerning theprocessing of tissue samples for quantitative PCR is available fromvarious sources, including manufacturers and vendors of commercialinstruments and reagents for qRT-PCR (e.g., Qiagen (Valencia, Calif.)and Ambion (Austin, Tex.)). Instruments and systems for automatedperformance of qRT-PCR are commercially available and used routinely inmany laboratories. An example of a well-known commercial system is theApplied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems,Foster City, Calif.).

Once mRNA is isolated, the first step in gene expression measurement byRT-PCR is the reverse transcription of the mRNA template into cDNA,which is then exponentially amplified in a PCR reaction. Two commonlyused reverse transcriptases are avilo myeloblastosis virus reversetranscriptase (AMV-RT) and Moloney murine leukemia virus reversetranscriptase (MMLV-RT). The reverse transcription reaction typically isprimed with specific primers, random hexamers, or oligo(dT) primers.Suitable primers are commercially available, e.g., GeneAmp® RNA PCR kit(Perkin Elmer, Waltham, Mass.). The resulting cDNA product can be usedas a template in the subsequent polymerase chain reaction.

The PCR step is carried out using a thermostable DNA-dependent DNApolymerase. The polymerase most commonly used in PCR systems is aThermus aquaticus (Taq) polymerase. The selectivity of PCR results fromthe use of primers that are complementary to the DNA region targeted foramplification, i.e., regions of the cDNAs reverse transcribed from genesencoding proteins of interest. Therefore, when qRT-PCR is employed inthe present invention, primers specific to each engraftment marker geneare based on the cDNA sequence of the gene. Commercial technologies suchas SYBR® Green or TagMan® (Applied Biosystems, Foster City, Calif.) canbe used in accordance with the vendor's instructions. Messenger RNAlevels can be normalized for differences in loading among samples bycomparing the levels of housekeeping genes such as beta-actin or GAPDH.The level of mRNA expression can be expressed relative to any singlecontrol sample, or a pool of control samples or a commercially availableset of control mRNA.

Suitable primer sets for PCR analysis of expression of engraftmentmarker genes can be designed and synthesized by one of skill in the art,without undue experimentation. Alternatively, PCR primer sets forpracticing the present invention can be purchased from commercialsources, e.g., Applied Biosystems. PCR primers preferably are about 17to 25 nucleotides in length. Primers can be designed to have aparticular melting temperature (Tm), using conventional algorithms forTm estimation. Software for primer design and Tm estimation areavailable commercially, e.g., Primer Express™ (Applied Biosystems), andalso are available on the internet, e.g., Primer3 (MassachusettsInstitute of Technology). By applying established principles of PCRprimer design, a large number of different primers can be used tomeasure the expression level of any given engraftment marker gene.

In some embodiments, RNA analysis is performed using a technology thatdoes not involve RNA extraction or isolation. One such technology isquantitative nuclease protection assay, which is commercially availableunder the name gNPA™ (High Throughput Genomics, Inc., Tucson, Ariz.).

The methods of the invention encompass “detecting expression” ofpositive engraftment markers as well as negative engraftment markers. Inthose embodiments involving detecting positive engraftment markers(whose expression positively correlates with the expression of other HVPmarkers, such as Isl1), “detecting expression” of the at least onepositive engraftment marker comprises detecting expression of mRNAencoding the at least one engraftment marker in the HVPs (detection of“positive” marker expression, such as a positive angiogenic marker(s)and/or a positive extracellular matrix marker(s)). Detecting expressionof mRNA encoding the positive engraftment marker(s) is intended toencompass direct assaying of the mRNA as well as indirect assaying ofthe mRNA, such as by assaying of cDNA prepared from the mRNA.

In those embodiments involving detecting negative engraftment markers(whose expression negatively correlates with the expression of other HVPmarkers, such as Isl1), “detecting expression” of the at least onenegative engraftment marker comprises detecting lack of expression ofmRNA encoding the at least one engraftment marker in the HVPs (detectionof lack of expression of “negative” markers, such as a negativeangiogenic marker(s) and/or a negative extracellular matrix marker(s)).Detecting lack of expression of mRNA encoding the negative engraftmentmarker(s) is intended to encompass direct assaying of the mRNA as wellas indirect assaying of the mRNA, such as by assaying of cDNA preparedfrom the mRNA. Furthermore, as used herein, “lack of expression” of anmRNA (e.g., an mRNA for a negative marker) is intended to mean that thePearson correlation index of expression relative to expression of apositive HVP marker, such as Islet 1, JAG1, FZD4, LIFR, FGFR3 and/orTNFSF9, is about −0.50 or less.

In certain embodiments, the methods of the invention can comprise (i)detecting expression of at least one positive engraftment marker (e.g.,positive angiogenic marker(s) and/or positive extracellular matrixmarker(s)); and (ii) detecting lack of expression of at least onenegative engraftment marker (e.g., negative angiogenic marker(s) and/ornegative extracellular matrix marker(s)). For example, in oneembodiment, the invention provides a method for engrafting humanventricular tissue in a subject, the method comprising:

-   -   culturing human cells containing cardiac progenitor cells under        conditions causing differentiation into human ventricular        progenitor cells (HVPs) expressing at least one surface marker        selected from the group consisting of JAG1, FZD4, LIFR, FGFR3        and/or TNFSF9;    -   isolating the HVPs expressing the at least one surface marker to        form an HVP population;    -   detecting expression of at least one positive engraftment marker        in a sample of the HVP population;    -   detecting lack of expression of at least one negative        engraftment marker in the sample of the HVP population; and    -   transplanting the HVPs expressing the at least one surface        marker and the at least one positive engraftment marker, and        lacking expression of the at least one negative engraftment        marker, into the subject such that human ventricular tissue is        engrafted in the subject.

Likewise, the other methods of the invention described above cansimilarly comprise the combination of: (i) detection of at least onepositive engraftment marker (e.g., positive angiogenic marker(s) and/orpositive extracellular matrix marker(s)); and (ii) detection of lack ofexpression of at least one negative engraftment marker (e.g., negativeangiogenic marker(s) and/or negative extracellular matrix marker(s)).

As described in detail in Example 14, gene expression profiling has alsobeen performed on the Islet 1 negative (Isl1−) subpopulation of cellswithin the Day 6 HVP population to thereby further characterize theIsl1− subpopulation of Day 6 progenitor cells that are not suitable fortransplantation and engraftment. These studies determined that theIsl1−Day 6 subpopulation of cells express the following genes atsignificant levels (average of 2000 copies or more of mRNA): ACTB,MTRNR2L2, MALAT1, EEF1A1, KRT8, MTRNR2L8, KRT18, FN1, MTRNR2L1, TTN,GAPDH, YWHAZ, MTRNR2L9, RPL3, AHNAK, KCNQ1OT1, TUBB, SLC2A3, FTL,HSP90B1, KRT19, HSPA8, MYL6, RPLP0, BSG, COL3A1, TPM1, VCAN, ENO1, RPL4,ACTG1, MTRNR2L10, HMGN2, PRTG, TPI1, HMGB1, VIM, ATP5B, HSP90AB1, RPL7,CBX5, MYL7, SERPINH1, HNRNPK, SRRM2, PODXL, EEF2, SPARC, ACTC1, HUWE1,COL1A2, LINC00506, HSPA5, MDK, HNRNPC, HSP90AA1, RGS5, LAMC1, APLNR,UGDH-AS1 and RPS3A. This Isl1−Day 6 subpopulation of cells express thefollowing genes at high levels (average of 5000 copies or more of mRNA):ACTB, MTRNR2L2, MALAT1, EEF1A1, KRT8, MTRNR2L8, KRT18, FN1, MTRNR2L1,TTN, GAPDH and YWHAZ.

Accordingly, this gene expression profile information for the Isl1−Day 6subpopulation allows for further refinement of the selection process forchoosing engraftable HVPs for transplantation. In particular, cellswithin a differentiated (e.g., day 5-7) culture of cardiac progenitorcells (e.g., cultured as described below and in Examples 1 and 11) canbe analyzed for their gene expression profile and those cells that lackexpression of Islet 1 (Isl1−) but that do express at least one markerselected from ACTB, MTRNR2L2, MALAT1, EEF1A1, KRT8, MTRNR2L8, KRT18,FN1, MTRNR2L1, TTN, GAPDH, YWHAZ, MTRNR2L9, RPL3, AHNAK, KCNQ1OT1, TUBB,SLC2A3, FTL, HSP90B1, KRT19, HSPA8, MYL6, RPLP0, BSG, COL3A1, TPM1,VCAN, ENO1, RPL4, ACTG1, MTRNR2L10, HMGN2, PRTG, TPI1, HMGB1, VIM,ATP5B, HSP90AB1, RPL7, CBX5, MYL7, SERPINH1, HNRNPK, SRRM2, PODXL, EEF2,SPARC, ACTC1, HUWE1, COL1A2, LINC00506, HSPA5, MDK, HNRNPC, HSP90AA1,RGS5, LAMC1, APLNR, UGDH-AS1 and RPS3A, can be ruled out fortransplantation. Alternatively, cells that lack expression of Islet 1(Isl1−) but that do express at least one marker selected from ACTB,MTRNR2L2, MALAT1, EEF1A1, KRT8, MTRNR2L8, KRT18, FN1, MTRNR2L1, TTN,GAPDH and YWHAZ can be ruled out for transplantation. In certainembodiments, cells ruled out for transplantation are Isl1− and expresstwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, fifteen or moreor twenty or more of the aforementioned markers.

Generation of Human Ventricular Progenitors (HVPs)

Human pluripotent stem cells can be cultured under conditions that leadto the generation of a non-clonal population of human ventricularprogenitor cells. Culture conditions for generating cardiac progenitorcells have been described in the art (see e.g., Lian, X. et al. (2012)Proc. Natl. Acad. Sci. USA 109:E1848-1857; U.S. Patent Publication No.20130189785) and also are described in detail in Example 1 and thedrawing, as well as in Example 11. Typically, Wnt/β-catenin signaling isfirst activated in the hPSCs, followed by an incubation period, followedby inhibition of Wnt/β-catenin signaling.

Activation of Wnt/β-catenin signaling is achieved by incubation with aGsk3 inhibitor, preferably CHIR98014 (CAS 556813-39-9). Inhibition ofWnt/β-catenin signaling is achieved by incubation with a Porcninhibitor, preferably Wnt-059 (CAS 1243243-89-1). The Gsk3 inhibitor isused to promote cardiac mesodermal differentiation, whereas the Porcninhibitor is used to enhance ventricular progenitor differentiation frommesoderm cells. With regard to timing of the use of the Gsk3 and Porcninhibitors, typically, at day 0 of culture, the hPSCs are cultured withthe Gsk3 inhibitor, at day 3 of culture the medium is changed to removethe Gsk3 inhibitor and the cells are then cultured with media containingthe Porcn inhibitor through day 5 of culture. HVP generation is optimalbetween days 5 and 7 (inclusive) in culture and peaks at day 6 ofculture. Other non-limiting, exemplary details on culture conditions andtiming of the use of the Gsk3 and Porcn inhibitors are described indetail in Examples 1 and 11.

Suitable hPSCs for use in the methods of the invention include inducedpluripotent stem cells (iPSC), such as 19-11-1, 19-9-7 or 6-9-9 cells(Yu, J. et al. (2009) Science 324:797-801), and human embryonic stemcell lines, such as ES03 cells (WiCell Research Institute) or H9 cells(Thomson, J. A. et al. (1998) Science 282:1145-1147). Suitable culturemedia for generating cardiomyogenic progenitors include E8 medium,mTeSR1 medium and RPMI/B27 minus insulin, each described further inExample 1 and/or Example 11.

Culture conditions have now been determined that bias the cardiomyogenicprogenitor cells to the ventricular lineage. These ventricularcardiomyogenic progenitor cells can be cultured in RPMI/B27 medium andthey can further differentiate into ventricular muscle cells. Apreferred medium for culturing the cardiac ventricular progenitor cellsin vitro such that they differentiation into ventricular cells in vitro(e.g., expressing the MLC2v marker described below) is the CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 20%KnockOut Serum Replacement, 2.5 mM GlutaMAX and 100 μg/ml Vitamin C).

Known markers of differentiated cardiac cells can be used to identifythe type(s) of cells that are generated by differentiation of thecardiac progenitor cells. For example, cardiac troponin I (cTnI) can beused as a marker of cardiomyocyte differentiation. CD144 (VE-cadherin)can be used as a marker of endothelial cells. Smooth muscle actin (SMA)can be used as a marker of smooth muscle cells. MLC2v can be used as amarker of ventricular muscle cells. MLC2a, which is expressed on bothimmature ventricular muscle cells and atrial muscle cells, can be usedas a marker for those cell types. Additionally, sarcolipin, which isspecifically expressed in atrial muscle cells, can be used as a markerfor atrial muscle cells. Phospholamban, which is expressed predominantlyin the ventricles and, to a lesser extent, in the atria, can also beused as a marker. Hairy-related transcription factor 1 (HRT1), alsocalled Hey1, which is expressed in atrial cardiomyocytes, can be used asa marker for atrial cardiomyocytes. HRT2 (Hey2), which is expressed inventricular cardiomyocytes, can be used as a marker for ventricularcardiomyocytes. In addition, IRX4 has a ventricular-restrictedexpression pattern during all stages of development, and thus can beused as a ventricular lineage marker. In summary, the genes expressed inthe ventricles, and thus which are appropriate ventricular markers, are:MLC2v, IRX4 and HRT2, while genes expressed in the atria, and thus whichare appropriate atrial markers are: MLC2a, HRT1, Sarcolipin and ANF(atrial natriuretic factor). The preferred marker of ventriculardifferentiation is MLC2v.

Methods of Isolating Human Cardiac Ventricular Progenitor Cells

Methods of isolating HVPs based on cell surface marker expression (e.g.,JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9) is described in detail in U.S.Ser. No. 14/832,324, filed Aug. 21, 2015, and U.S. Ser. No. 14/984,783,filed Dec. 30, 2015, the entire contents of each of which are expresslyincorporated herein by reference). In brief, agents reactive with thecell surface marker are used to isolate the HVPs according to procedureswell established in the art. Identification of JAG1, FZD4, LIFR andFGFR3 as cell surface markers of HVPs is also described in detail inExamples 2, 4 and 5.

In one embodiment, the agent reactive with the cell surface marker is anantibody that specifically binds to the cell surface marker, such as amonoclonal antibody. Non-limiting examples include murine, rabbit,human, humanized or chimeric monoclonal antibodies with bindingspecificity for the cells surface marker (e.g., anti-JAG1, anti-FZD4,anti-LIFR, anti-FGFR3 and/or anti-TNFSF9 antibodies). Such monoclonalantibodies are commercially available in the art (e.g., R&D Systems,Santa Cruz Biotechnology). Moreover, such antibodies can be preparedusing standard techniques well established in the art using the cellsurface marker as the antigen.

In another embodiment, the agent reactive with the cell surface markeris a ligand for the cell surface marker, such as a soluble ligand or asoluble ligand fusion protein (e.g., an Ig fusion protein). Solubleligands can be prepared using standard recombinant DNA techniques, forexample by deletion of the transmembrane and cytoplasmic domains. Asoluble ligand can be transformed into a soluble ligand fusion proteinalso using standard recombinant DNA techniques. A fusion protein can beprepared in which fusion partner can comprise a binding moiety thatfacilitates separation of the fusion protein.

In order to separate the cell surface marker positive cells fromnon-reactive cells, one of a variety of different cell separationtechniques known in the art can be used. Preferably, the cell surfacemarker positive cells are separated from non-reactive cells byfluorescence activated cell sorting (FACS). The FACS technology, andapparatuses for carrying it out to separate cells, is well establishedin the art. When FACS is used for cell separation, preferably theagent(s) reactive with the cell surface marker that is used is afluorescently-labeled monoclonal antibody. Alternatively, cellseparation can be achieved by, for example, magnetic activated cellsorting (MACS). When MACS is used for cell separation, preferably theagent reactive with the cell surface marker that is used is magneticnanoparticles coated with monoclonal antibody. Alternatively, othersingle cell sorting methodologies known in the art can be applied to themethods of isolating human ventricular progenitor cells of theinvention, including but not limited to IsoRaft array and DEPArraytechnologies.

Clonal Populations of Human Cardiac Ventricular Progenitor Cells

Clonal populations of HVPs can be obtained by expansion and propagationof the HVPs such that a clonal population of a billion or more cells canbe achieved. The ability to clonally expand the human ventricularprogenitor cells to such large numbers is a necessary feature forsuccessful use of these cells in vivo to enhance cardiac function, sincesuch a use requires on the order of a billion or more cells. Asdescribed further in the Examples, such a single cell can be obtained atapproximately day 6 of the culture under conditions that promote thegeneration of cardiomyogenic progenitors. The clonal population of humancardiac ventricular progenitors can be further cultured anddifferentiated in vitro such that the cells express the ventricularmaker MLC2v. Preferably, the single human cardiac ventricular progenitorcell is isolated by fluorescence activated cell sorting. Alternatively,the cell can be isolated by MACS or by other cell sorting methods knownin the art and/or described herein. Preferably, the single human cardiacventricular progenitor cell is cultured in Cardiac Progenitor Culture(CPC) medium, as described herein (see e.g., Example 3).

Pharmaceutical compositions comprising the clonal population of cardiacventricular progenitor cells can be prepared by standard methods knownin the art. The pharmaceutical compositions typically are sterile andcan comprise buffers, media, excipients and the like suitable forpharmaceutical administration. In one embodiment, the pharmaceuticalcomposition comprising the clonal population is formulated onto a threedimensional (3D) matrix. Compositions formulated onto a 3D matrix areparticularly preferred for formation of a heart muscle cell patch thatcan be transplanted in vivo for heart muscle repair. Furthermore, thecompositions can be formulated into two dimensional (2D) sheets ofcells, such as a muscular thin film (MTF) as described in Domian, I. J.et al. (2009) Science 326:426-429. Such 2D sheets of cell tissue alsocan be used in the formation of a heart muscle cell patch that can betransplanted in vivo for heart muscle repair.

In Vivo Tissue Engineering

In vivo transplantation studies, described in Example 7 and 8 in whichthe human ventricular progenitors (HVPs) were transplanted under thekidney capsule in nude mice, document the ability of the HVPs tospontaneously assemble into a large wall of mature, functional, humanventricular muscle on the surface of the kidney capsule. Vascularizationoccurs via a paracrine pathway by calling the murine vasculature to theventricular muscle wall, while a matrix is generated via a cellautonomous pathway from the progenitors themselves. In vivointra-myocardial transplantation studies described in Example 9 in whichthe HVPs were transplanted into the normal murine heart document thatthe HVPs spontaneously migrate to the epicardial surface, where theyexpand, subsequently differentiate, and mature into a wall of humanventricular muscle on the surface of the epicardium. Taken together,these studies show that human ventriculogenesis can occur via acompletely cell autonomous pathway in vivo via purified HVPs, therebyallowing their use in organ-on-organ in vivo tissue engineering.

The human ventricular myocardium has a limited capacity forregeneration, most of which is lost after 10 years of age (Bergmann, O.et al. (2015) Cell 161:1566-1575). As such, new strategies to generateheart muscle repair, regeneration, and tissue engineering approachesduring cardiac injury have been a subject of intense investigation inregenerative biology and medicine (Sahara, M. et al. (2015) EMBO J.34:710-738; Segers, V. F. M. and Lee, R. T. (2008) Nature 451:937-942).Given the need to achieve coordinated vascularization and matrixformation during tissue engineering of any solid organ, the assumptionhas been that the formation of an intact 3-D solid organ in vivo willultimately require the addition of vascular cells and/or conduits, aswell as biomaterials and/or decellularized matrix that will allowalignment and the generation of contractile force (Forbes, S. J. andRosenthal, N. (2014) Nature Med. 20:857-869; Harrison, R. H. et al.(2014) Tissue Eng. Part B Rev. 20:1-16). The complexity of adding thesevarious components to achieve the formation of a functional solid organhas confounded attempts to reduce this to clinical practice (Webber, M.J. et al. (2014) Ann. Biomed. Eng. 43:641-656). Although hPSCs holdgreat promise, to date, it has not been possible to build a pure,vascularized, fully functional, and mature 3-D human ventricular muscleorgan in vivo on the surface of a heart in any mammalian system(Vunjak-Novakovic, G. et al. (2011) Annu. Rev. Biomed. Eng. 13:245-267).

The ability of generate billions of purified HVPs from a renewablesource of either human ES or iPS cell lines, and detect engraftmentmarkers indicative of the ability of the cells to engraft in vivo,represents a new approach to the generation of functional ventricularmuscle in the setting of advanced heart failure. The progenitors can bedelivered by intramyocardial injection and then self-migrate to theepicardial surface where they expand and differentiate, losingprogenitor markers. Over the course of several week, the cells exit thecell cycle, and proceed to form adult rod-shaped cells that displayseveral independent markers of mature ventricular myocardium includingthe formation of T tubules, catecholamine responsiveness, loss ofautomaticity, adult rod shaped conformation with aligned sarcomericstructures, and the ability to generate force that is comparable toother heart muscle patches derived from hPSCs differentiatedcardiomyocytes (Tulloch, N. L. et al. (2011) Circ. Res. 109:47-59). Thescalability of this cell autonomous pathway has allowed the ectopicgeneration of human ventricular muscle that has a combined thickness inexcess of 1.5 cm in thickness, approaching levels that correspond to thehuman ventricular free wall (Basavarajaiah, S. et al. (2007) Br. J.Sports Med. 41:784-788).

The ability to migrate to the epicardial niche, the site of most of theadult heart progenitors at later stages, is a unique feature of HVPs,and mimics the normal niche of these cells during expansion of theventricular compact zone during ventriculogenesis. Previous studies haveshown that the generation of acute ischemic injury and a breakdown invascular permeability are a pre-requisite for the grafting of relativelysmall numbers of ES cell derived cardiomyocytes into injured myocardium(van Laake, L. W. et al. (2007) Stem Cell Res. 1:9-24; Laflamme, M. A.et al. (2007) Nat. Biotechnol. 25:1015-1024), and even then the survivalrate is low (<5%) (Laflamme, M. A. and Murry, C. E. (2011) Nature473:326-335; Laflamme, M. A. et al. (2005) Am. J. Pathol. 167:663-671).The ability of intra-myocardial HVPs to form an extensive ventricularpatch on the epicardial surface in the absence of acute ischemic injuryprovides a new therapeutic strategy for dilated cardiomyopathy withoutthe need for additional biomaterials, cells, or transfer of exogenousgenes and/or RNAs.

The ability to form a 3-D ventricular muscle wall on the epicardialsurface of the in vivo normal heart is a unique feature of theISL1/FZD4/JAG1/LIFR/FGFR3/TNFSF9 ventricular progenitors as later stageprogenitors do not display the ability for the formation ofthree-dimensional ventricular tissue in either the cardiac ornon-cardiac context, emphasizing the importance of generating acommitted ventricular lineage as well as purifying the specificventricular progenitor at a specific stage of ventriculogenesis.

Accordingly, the invention provides methods for generating humanventricular tissue in vivo using the HVPs described herein. In oneembodiment, the method comprises transplanting engraftable HVPs into anorgan of a non-human animal wherein the engraftable HVPs (i) express atleast one cell surface marker selected from the group consisting ofJAG1, FZD4, LIFR, FGFR3 and TNFSF9; and (ii) express at least oneengraftment marker; and allowing the HVPs to grow in vivo such thathuman ventricular tissue is generated. Preferably, the non-human animalis immunodeficient such that it cannot mount an immune response againstthe human progenitor cells. In one embodiment, the non-human animal is amouse, such as an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mouseor an immunodeficient SCID-beige mouse (commercially available fromCharles River France). In one embodiment, the organ is a kidney (e.g.,the cells are transplanted under the kidney capsule). In anotherembodiment, the organ is a heart. In various embodiments, at least 1×10⁶cells, at least 2×10⁶ cells, at least 3×10⁶ cells, at least 4×10⁶ cells,at least 5×10⁶ cells, at least 1×10⁷ cells, at least 5×10⁷ cells, atleast 1×10⁸ cells, at least 1×10⁹ cells are transplanted.

To obtain engraftable HVPs for transplantation, human pluripotent stemcells (hPSCs) can be cultured in vitro under conditions leading to thegeneration of HVPs, as described herein (referred to herein as the HVPGprotocol), followed by detection of the requisite cell surface marker(s)and engraftment marker(s).

Regarding the timing of transplanting HVPs post in-vitro culture, foroptimal ventricular tissue generation the cells should be transplantedat a stage that can be defined based on the cellular markers expressedby the HVPs at the time of transplantation, determined at days post thestart of culture, which is defined as day 0 of the HVPG protocol. In oneembodiment, the cells are transplanted after the peak of cardiacmesoderm formation, which can be defined as peak expression of themesodermal marker MESP1. Typically, MESP1 expression is between day 2and day 4 of culture (inclusive) and peaks at day 3. In one embodiment,the cells are transplanted at the time corresponding to peak Islet-1expression. Typically, Islet 1 is expressed between day 4 to day 8 ofculture (inclusive) and peaks at day 6 of culture. In one embodiment,the cells are transplanted before the peak of NKX2.5 expression.Typically, NKX2.5 expression starts at day 6 of culture, peaks at day 10of culture and is then maintained afterwards. In one embodiment, thecells are transplanted prior to the peak expression of the downstreamgenes MEF-2 and TBX-1. Typically, these downstream genes are expressedbetween day 5 and day 15 of culture (inclusive) and peaks at day 8 ofculture. In one embodiment, the cells are transplanted prior to theexpression of differentiated contractile protein genes. Typically, theexpression of contractile protein genes (including TNNT2 and MYH6)starts from day 10 of culture onward. In certain embodiments, the cellsare transplanted at a time when two, three or four of the aforementionedmarker patterns are present. In another embodiment, the cells aretransplanted at a time when all five of the aforementioned markerpatterns are present. In one embodiment, the cells are transplantedbetween day 4 to day 8 (inclusive) of culture. In a more preferredembodiment, the cells are transplanted between day 5 to day 7(inclusive) of culture. In the most preferred embodiment, the cells aretransplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue. In various embodiments,the cells are allowed to grow for one week, two weeks, one month, twomonths, three months, four months, five months or six months. The methodcan further comprise harvesting ventricular tissue from the non-humananimal after growth of the cells and differentiation into ventriculartissue.

Methods of Enhancing Cardiac Function

The methods of the invention for identifying and engrafting engraftableHVPs can be used in vivo to enhance cardiac function by transplantingthe cells directly into the heart. It has now been shown that the HVPshave the capacity to differentiate into all three types of cardiaclineage cells (cardiac myocytes, endothelial cells and smooth musclecells) (see Example 3). Furthermore, when cultured under conditions thatbias toward the ventricular lineage, the HVPs have been shown to adopt apredominantly ventricular muscle phenotype when transplanted into thenatural ventricle environment in vivo, demonstrating that theseprogenitor cells “recognize” the ventricular environment and respond anddifferentiate appropriately in vivo. Since damage to the ventricularenvironment is largely responsible for the impaired cardiac function incardiac diseases and disorders, the ability to restore ventricularmuscle cells using the ventricular progenitor cells of the inventionrepresents a significant advance in the art.

To enhance cardiac function, preferably a clonal population of HVPs(e.g., in a pharmaceutical composition) is administered directly intothe heart of the subject. More preferably, the clonal population isadministered directly into a ventricular region of the heart of thesubject. In one embodiment, the pharmaceutical composition administeredto the subject comprises the clonal population formulated onto a threedimensional matrix.

The methods of the invention for enhancing cardiac function in a subjectcan be used in a variety of clinical situations involving damage to theheart or reduced or impaired cardiac function, such as in cardiovascularconditions, diseases and disorders. Non-limiting examples of suchclinical situations include a subject who has suffered a myocardialinfarction and a subject who has a congenital heart disorder. Examplesof preferred cardiovascular conditions, diseases or disorders includecoronary artery disease and acute coronary syndrome.

Methods of Use of Cardiac Ventricular Progenitor Cells In Vitro

The cardiac ventricular progenitor cells of the invention identified asexpressing at least one cell surface marker (JAG1, FZD4, LIFR, FGFR3and/or TNFSF9), as well as at least one engraftment marker (angiogenicmarkers and/or extracellular matrix markers) can be used in vitro in thestudy of various aspects of cardiac maturation and differentiation, inparticular in identifying the cells signaling pathways and biologicalmediators involved in the process of cardiac maturation anddifferentiation.

Furthermore, since cardiac ventricular progenitor cells are committed tothe cardiac lineage and, moreover, are biased toward ventriculardifferentiation, these progenitor cells also are useful for evaluatingthe cardiac toxicity of test compounds. All potential new drugs andtherapeutics must be evaluated for their toxicity to cardiac cells,before they can be deemed safe for use in humans. Thus, the ability toassess cardiac toxicity in an in vitro culture system is veryadvantageous. Accordingly, in another aspect, the invention provides amethod of screening for cardiac toxicity of test compound, the methodcomprising

Providing human cardiac ventricular progenitor cells that (i) express atleast one cell surface marker selected from the group consisting ofJAG1, FZD4, LIFR, FGFR3 and TNFSF9; and (ii) express at least oneengraftment marker;

contacting the cells with the test compound; and

measuring toxicity of the test compound for the cells,

wherein toxicity of the test compound for the cells indicates cardiactoxicity of the test compound.

In a preferred embodiment, the HVPs are provided by isolating the cellsaccording to the methods described herein. In a particularly preferredembodiment, the cells are isolated by separating cell surface markerexpressing cells from a cell culture comprising cardiac progenitor cellsusing an antibody that specifically binds to the cell surface marker.Preferably, the cells are isolated using FACS or MACS as describedherein. In yet another embodiment, the HVPs are further cultured anddifferentiation into MLC2v+ ventricular cells prior to contacting withthe test compound.

The toxicity of the test compound for the cells can be measured by oneor more of a variety of different methods for assessing cell viabilityor other physiological functions. Preferably, the effect of the testcompound on cell viability is measured using a standard cell viabilityassay, wherein reduced cell viability in the presence of the testcompound is indicative of cardiac toxicity of the test compound.Additionally or alternatively, cell growth can be measured. Additionallyor alternatively, other indicators of physiological functions can bemeasured, such as cell adhesion, cell signaling, surface markerexpression, gene expression and the like. Similarly, a negative effectof the test compound on any of these indicators of physiologicalfunction is indicative of cardiac toxicity of the test compound.

The invention further provides a method of identifying a compound thatmodulates human cardiac ventricular progenitor cell differentiation, themethod comprising

providing human cardiac ventricular progenitor cells that (i) express atleast one cell surface marker selected from the group consisting ofJAG1, FZD4, LIFR, FGFR3 and TNFSF9; and (ii) express at least oneengraftment marker;

culturing the cells in the presence or absence of a test compound;

measuring differentiation of the cells in the presence or absence of thetest compound; and

selecting a test compound that modulates human cardiac ventricularprogenitor cell differentiation, as compared to differentiation in theabsence of the test compound, to thereby identify a compound thatmodulates human cardiac ventricular progenitor cell differentiation.

In one embodiment, the test compound stimulates human cardiacventricular progenitor cell differentiation. In another embodiment, thetest compound inhibits human cardiac ventricular progenitor celldifferentiation. Differentiation of the cells can be measured by, forexample, measurement of the expression of differentiation markersappearing on the cultured cells over time, as described herein. In apreferred embodiment, the HVPs are provided by isolating the cellsaccording to the methods described herein. In a particularly preferredembodiment, the cells are isolated by separating cell surface markerexpressing cells from a cell culture comprising cardiac progenitor cellsusing an antibody that specifically binds to the cell surface marker.Preferably, the cells are isolated using FACS or MACS as describedherein.

The invention further provides a method of identifying a compound thatmodulates human ventricular cardiomyocyte function, the methodcomprising

providing human cardiac ventricular progenitor cells that (i) express atleast one cell surface marker selected from the group consisting ofJAG1, FZD4, LIFR, FGFR3 and TNFSF9; and (ii) express at least oneengraftment marker;

culturing the cells in the presence or absence of a test compound underconditions that generate human ventricular cardiomyocytes;

measuring function of the human ventricular cardiomyocytes in thepresence or absence of the test compound; and

selecting a test compound that modulates human ventricular cardiomyocytefunction, as compared to function in the absence of the test compound,to thereby identify a compound that modulates human ventricularcardiomyoctye function.

In one embodiment, the test compound stimulates human ventricularcardiomyocyte function. In another embodiment, the test compoundinhibits human ventricular cardiomyocyte function. Function of the cellscan be measured by measurement of any suitable indicator of ventricularcell function, including but not limited to, for example, formation of Ttubules, acquisition of adult-rod shaped ventricular cardiomyocytes, andability to generate force in response to electrical stimulation.Suitable assays for measuring such indicators of ventricular cellfunction are known in the art. In a preferred embodiment, the HVPs areprovided by isolating the cells according to the methods describedherein. In a particularly preferred embodiment, the cells are isolatedby separating cell surface marker expressing cells from a cell culturecomprising cardiac progenitor cells using an antibody that specificallybinds to the cell surface marker. Preferably, the cells are isolatedusing FACS or MACS as described herein.

In Vivo Animal Models Using Human Ventricular Progenitor Cells

The development of human iPS and ES cell based models of cardiac diseasehas opened new horizons in cardiovascular drug development anddiscovery. However, to date, these systems have had the limitations ofbeing based on 2D structures in cultured cell systems. In addition, thefetal and immature properties of the cells limit their utility andfidelity to the adult heart. Human cardiac disease, in particular heartfailure, is a complex, multifactorial, multi-organ disease, that isinfluenced by environmental, hormonal, and other key organs that areknown sites for therapeutic targets, such as the kidney. The ability tobuild a mature functional human ventricular organ either ectopically oron the surface of the intact normal murine heart opens up a new in vivomodel system to allow studies that normally could only be assayed on amature human ventricular muscle chamber, such as ventriculararrhythmias, generation of contractile force, fibrosis, and thepotential for regeneration. Accordingly, the option to study humancardiac disease outside of the in vitro tissue culture systems, anddirectly in the context of heart failure in vivo, is now clearlypossible.

Thus, the engraftable HVPs identified according to the methods of theinvention also can be used to create animal models that allow for invivo assessment of human cardiac tissue function and for in vivoscreening of compounds, such as to determine the cardiac toxicity of atest compound in vivo or to identify compounds that modulate humancardiac tissue differentiation or function in vivo. Accordingly, theinvention provides methods for testing the effects of test compounds onhuman ventricular tissue in vivo using the HVPs described herein. In oneembodiment, the method comprises:

transplanting engraftable HVPs into an organ of a non-human animalwherein the engraftable HVPs (i) express at least one cell surfacemarker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9; and (ii) express at least one engraftment marker;

allowing the progenitors to grow in vivo such that human ventriculartissue is generated;

administering a test compound to the non-human animal; and

evaluating the effect of the test compound on the human ventriculartissue in the non-human animal.

In another embodiment, the method comprises:

administering a test compound to a non-human animal, wherein thenon-human animal comprises engraftable human ventricular progenitors(HVPs) transplanted into an organ of the non-human animal, wherein theengraftable HVPs (i) express at least one cell surface marker selectedfrom the group consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9; and(ii) express at least one engraftment marker; and

evaluating the effect of the test compound on the engraftable HVPs inthe non-human animal.

In one embodiment, the cardiac toxicity of the test compound isevaluated, for example by measuring the effect of the test compound onthe viability of the human ventricular tissue or the human ventricularprogenitors in the non-human animal (as compared to the viability of thetissue or progenitors in the absence of the test compound). Cellviability can be assessed by standard methods known in the art.

In another embodiment, the ability of a test compound to modulatecardiac differentiation can be evaluated, for example by measuring theeffect of the test compound on the differentiation of the humanventricular tissue or HVPs in the non-human animal (as compared to thedifferentiation of the tissue or progenitors in the absence of the testcompound). Differentiation of the cells can be measured by, for example,measurement of the expression of differentiation markers appearing onthe cells over time.

In another embodiment, the ability of a test compound to modulatecardiac function can be evaluated, for example by measuring the effectof the test compound on the function of the human ventricular tissue orHVPs in the non-human animal (as compared to the function of the tissueor HVPs in the absence of the test compound). Function of the tissue orHVPs can be measured by measurement of any suitable indicator ofventricular cell function, including but not limited to, for example,formation of T tubules, acquisition of adult-rod shaped ventricularcardiomyocytes, and ability to generate force in response to electricalstimulation. Suitable assays for measuring such indicators ofventricular cell function are known in the art.

Preferably, the non-human animal is immunodeficient such that it cannotmount an immune response against the human progenitor cells. In oneembodiment, the non-human animal is a mouse, such as an immunodeficientNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mouse or an immunodeficient SCID-beigemouse (commercially available from Charles River France). In oneembodiment, the organ is a kidney (e.g., the cells are transplantedunder the kidney capsule). In another embodiment, the organ is a heart.In various embodiments, at least 1×10⁶ cells, at least 2×10⁶ cells, atleast 3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least1×10⁷ cells, at least 5×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹cells are transplanted.

To create the animal models, engraftable HVPs for transplantation can beobtained as described above by culturing of hPSCs in vitro underconditions leading to the generation of HVPs, followed by detection ofthe requisite cell surface marker(s) and engraftment marker(s).Regarding the timing of transplanting HVPs post in-vitro culture, foroptimal ventricular tissue generation the cells should be transplantedat a stage that can be defined based on the cellular markers expressedby the HVPs at the time of transplantation, determined at days post thestart of culture, which is defined as day 0 of the HVPG protocol. In oneembodiment, the cells are transplanted after the peak of cardiacmesoderm formation, which can be defined as peak expression of themesodermal marker MESP1. Typically, MESP1 expression is between day 2and day 4 of culture (inclusive) and peaks at day 3. In one embodiment,the cells are transplanted at the time corresponding to peak Islet-1expression. Typically, Islet 1 is expressed between day 4 to day 8 ofculture (inclusive) and peaks at day 6 of culture. In one embodiment,the cells are transplanted before the peak of NKX2.5 expression.Typically, NKX2.5 expression starts at day 6 of culture, peaks at day 10of culture and is then maintained afterwards. In one embodiment, thecells are transplanted prior to the peak expression of the downstreamgenes MEF-2 and TBX-1. Typically, these downstream genes are expressedbetween day 5 and day 15 of culture (inclusive) and peaks at day 8 ofculture. In one embodiment, the cells are transplanted prior to theexpression of differentiated contractile protein genes. Typically, theexpression of contractile protein genes (including TNNT2 and MYH6)starts from day 10 of culture onward. In certain embodiments, the cellsare transplanted at a time when two, three or four of the aforementionedmarker patterns are present. In another embodiment, the cells aretransplanted at a time when all five of the aforementioned markerpatterns are present. In one embodiment, the cells are transplantedbetween day 4 to day 8 (inclusive) of culture. In a more preferredembodiment, the cells are transplanted between day 5 to day 7(inclusive) of culture. In the most preferred embodiment, the cells aretransplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue, prior to administrationof the test compound(s). In various embodiments, the cells are allowedto grow for one week, two weeks, one month, two months, three months,four months, five months or six months.

Kits

In another aspect, the invention provides kits for preparing engraftableHVPs. In one embodiment, the kit comprises means for isolating HVPsexpressing at least one cell surface marker selected from the groupconsisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9 and means for detectingat least one engraftment marker expressed by the isolated HVPs, alongwith instructions for use of the kit to isolate engraftable HVPs. In oneembodiment, the kit comprises means for detecting at least oneangiogenic engraftment marker (positive and/or negative angiogenicmarker(s)). In another embodiment, the kit comprises means for detectingat least one extracellular matrix engraftment marker (positive and/ornegative extracellular matrix marker(s)). In yet another embodiment, thekit comprises means for detecting at least one angiogenic engraftmentmarker and at least one extracellular matrix engraftment marker. Meansfor detecting any of the engraftment markers described above withrespect to the methods of identifying and transplanting engraftable HVPscan be included in a kit of the invention. Preferred angiogenic markersfor detection in a kit of the invention include FGF10, PRKD1, CCBE1,PDGFRA, EPHB2, GATA2 and NTRK1. Preferred extracellular matrix markersfor detection in a kit of the invention include FGF10, SMOC1 and CCBE1.

In one embodiment, the means for isolating the HVPS expressing at leastone cell surface marker selected from the group consisting of JAG1,FZD4, LIFR, FGFR3 and TNFSF9 is one or more agents reactive with the oneor more of the cell surface marker(s). In one embodiment, the agent(s)reactive with the cell surface marker(s) is an antibody thatspecifically binds to the cell surface marker, such as a monoclonalantibody. Non-limiting examples of such agents include murine, rabbit,human, humanized or chimeric monoclonal antibodies with bindingspecificity for the cells surface marker (e.g., anti-JAG1, anti-FZD4,anti-LIFR, anti-FGFR3 and/or anti-TNFSF9 antibodies). Such monoclonalantibodies are commercially available in the art (e.g., R&D Systems,Santa Cruz Biotechnology). Moreover, such antibodies can be preparedusing standard techniques well established in the art using the cellsurface marker as the antigen. In another embodiment, the agent reactivewith the cell surface marker is a ligand for the cell surface marker,such as a soluble ligand or a soluble ligand fusion protein (e.g., an Igfusion protein). Soluble ligands can be prepared using standardrecombinant DNA techniques, for example by deletion of the transmembraneand cytoplasmic domains. A soluble ligand can be transformed into asoluble ligand fusion protein also using standard recombinant DNAtechniques. A fusion protein can be prepared in which fusion partner cancomprise a binding moiety that facilitates separation of the fusionprotein.

In one embodiment, the means for detecting the at least one engraftmentmarker is one or more nucleic acids that detect RNA (e.g., mRNA) or DNA(e.g., cDNA) encoding the engraftment marker(s). In one embodiment, thenucleic acid(s) that detects RNA or DNA encoding the engraftmentmarker(s) is an oligonucleotide probe capable of hybridizing to RNA orDNA encoding the engraftment marker(s). In another embodiment, thenucleic acid(s) that detects RNA or DNA encoding the engraftmentmarker(s) is a pair of oligonucleotide primers capable of amplifyingnucleic acid encoding the engraftment marker(s), for example PCRprimers. Such oligonucleotide probes and primers can be designed andprepared (e.g., synthesized) based on the publicly available nucleotidesequences of the engraftment markers disclosed herein using standardrecombinant DNA methodologies.

In another embodiment, the means for detecting the at least oneengraftment marker is one or more agents reactive with the engraftmentmarker protein. The agent(s) reactive with the engraftment markerprotein(s) can be, for example, an antibody (e.g., monoclonal antibodyor other suitable antibody as described above with respect to the agentsreactive with the cell surface markers) or a ligand (e.g., solubleligand or soluble ligand fusion protein, as described above with respectto the agents reactive with the cell surface markers). Suitableantibodies or ligand agents reactive with an engraftment marker proteinare known in the art and/or can be prepared using standard recombinantDNA techniques.

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents ofFIGURES and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1: Generation of Human Isl1+ Cardiomyogenic ProgenitorCells by Modulation of Wnt Signaling in Human Pluripotent Stem Cells

Temporal modulation of canonical Wnt signaling has been shown to besufficient to generate functional cardiomyocytes at high yield andpurity from numerous hPSC lines (Lian, X. et al. (2012) Proc. Natl.Acad. Sci. USA 109:E1848-1857; Lian, X. et al. (2013) Nat. Protoc.8:162-175). In this approach, Wnt/β-catenin signaling first is activatedin the hPSCs, followed by an incubation period, followed by inhibitionof Wnt/β-catenin signaling. In the originally published protocol,Wnt/β-catenin signaling activation was achieved by incubation with theGsk3 inhibitor CHIR99021 (GSK-3 α, IC₅₀=10 nM; GSK-3, β IC₅₀=6.7 nM) andWnt/β-catenin signaling inhibition was achieved by incubation with thePorcn inhibitor IWP2 (IC₅₀=27 nM). Because we used Gsk3 inhibitor andWnt production inhibitor for cardiac differentiation, this protocol wastermed GiWi protocol. To improve the efficiency of the original protocoland reduce the potential side effects of the small molecules used in theoriginal protocol, a second generation protocol was developed that usesanother set of small molecules with higher inhibition potency. In thissecond generation GiWi protocol, Wnt/β-catenin signaling activation wasachieved by incubation with the Gsk3 inhibitor CHIR98014 (CAS556813-39-9; commercially available from, e.g., Selleckchem) (GSK-3 α,IC₅₀=0.65 nM; GSK-3, β IC₅₀=0.58 nM) and Wnt/β-catenin signalinginhibition was achieved by incubation with the Porcn inhibitor Wnt-059(CAS 1243243-89-1; commercially available from, e.g., Selleckchem orTocris) (IC₅₀=74 pM). The Gsk3 inhibitor CHIR98014 was used to promotecardiac mesodermal differentiation, whereas the Porcn inhibitor Wnt-059was used to enhance ventricular progenitor differentiation from mesodermcells.

For cardiomyocyte differentiation via the use of these small molecules,hPSCs were maintained on Matrigel (BD Biosciences) coated plates(Corning) in E8 medium (described in Chen, G. et al. (2011) NatureMethods, 8:424-429; commercially available; STEMCELL Technologies) ormTeSR1 medium (commercially available; STEMCELL Technologies). SuitablehPSCs include induced pluripotent stem cells (iPSCs) such as 19-11-1,19-9-7 or 6-9-9 cells (Yu, J. et al. (2009) Science, 324:797-801) andhuman embryonic stem cells (hESCs), such as ES03 (WiCell ResearchInstitute) and H9 cells (Thomson, J. A. et al. (1998) Science,282:1145-1147).

hPSCs maintained on a Matrigel-coated surface in mTeSR1 medium weredissociated into single cells with Accutase (Life Technologies) at 37°C. for 5 minutes and then seeded onto a Matrigel-coated cell culturedish at 100,000-200,000 cells/cm² in mTeSR1 medium supplemented with 5μM ROCK inhibitor Y-27632 (Selleckchem) (day −2) for 24 hours. Cellswere then cultured in mTeSR1, changed daily. At day 0, cells were thentreated with 1 μM Gsk3 inhibitor CHIR98014 (Selleckchem) for 24 hours(day 0 to day 1) in RPMI/B27-ins (500 ml RPMI with 10 ml B27 supplementwithout insulin). The medium was then changed to the correspondingmedium containing 2 μM the Porcn inhibitor Wnt-059 (Selleckchem) at day3, which was then removed during the medium change on day 5. Cells weremaintained in RPMI/B27 (stock solution: 500 ml RMPI medium+10 ml B27supplement) starting from day 7, with the medium changed every threedays. This exemplary culturing protocol for generating cardiomyogenicprogenitor cells is illustrated schematically in the drawing.

Flow cytometry and immunostaining were preformed to examine theexpression of particular lineage markers. After 24 hour treatment withCHIR-98014, more than 99% of the hPSCs expressed the mesoderm markerBrachyury. Three days after treatment with CHIR-98014, more than 95% ofdifferentiated cells expressed Mesp1, which marks the cardiac mesoderm.The culture protocol not only allowed the cells to synchronouslydifferentiate into the cardiac mesodermal lineage, but also reproduciblygenerated more than 90% of ventricular myocytes after 14 days ofdifferentiation, as determined by cTnT flow cytometry andelectrophysiology analysis.

To further assess cardiac differentiation of the hPSCs over time,Western blot analysis was performed on days 0-7 and d11 to examine theexpression of Isl1 and Nkx2.5 (cardiomyogenic progenitor markers) andcTnI (a cardiac myocyte marker). Cells were lysed in M-PER MammalianProtein Extraction Reagent (Pierce) in the presence of Halt Protease andPhosphatase Inhibitor Cocktail (Pierce). Proteins were separated by 10%Tris-Glycine SDS/PAGE (Invitrogen) under denaturing conditions andtransferred to a nitrocellulose membrane. After blocking with 5% driedmilk in TBST, the membrane was incubated with primary antibody overnightat 4° C. The membrane was then washed, incubated with ananti-mouse/rabbit peroxidase-conjugated secondary antibody at roomtemperature for 1 hour, and developed by SuperSignal chemiluminescence(Pierce). During cardiac differentiation of hPSCs, Isl1 expressionstarted on day 4 and increased to its maximum expression on day 6,whereas NKx2.5 only started to express on day 6 and reached its maximumexpression after day 10. Cardiomyoctes (cTnI+ cells) were not induceduntil day 11 of differentiation.

In addition, immunostaining of the day 6 cells was performed for Isl1expression. Cells were fixed with 4% formaldehyde for 15 minutes at roomtemperature and then stained with primary (anti-Isl1) and secondaryantibodies in PBS plus 0.4% Triton X-100 and 5% non-fat dry milk(Bio-Rad). Nuclei were stained with Gold Anti-fade Reagent with DAPI(Invitrogen). An epifluorescence microscope (Leica DM IRB) with aQImaging® Retiga 4000R camera was used for imaging analysis. The resultsshowed substantial numbers of Isl1+ cells.

Flow cytometry analysis of day 6 cells for Isl1 expression also wasperformed. Cells were dissociated into single cells with Accutase for 10minutes and then fixed with 1% paraformaldehyde for 20 minutes at roomtemperature and stained with primary and secondary antibodies in PBS0.1% Triton X-100 and 0.5% BSA. Data were collected on a FACSCaliberflow cytometer (Beckton Dickinson) and analyzed using FloJo. The resultsshowed that more than 95% of cells expressed Isl1 at this stage.

In summary, this example provides a protocol for human ventricularprogenitor generation (HVPG protocol) that allows for the large-scaleproduction of billions of Isl1+ human HPVs efficiently within 6 days.

Example 2: Identification of Jagged 1 as a Cell Surface Marker of HumanVentricular Progenitor Cells

To profile the transcriptional changes that occur during the cardiacdifferentiation process at a genome-scale level, RNA sequencing(RNA-seq) was performed at different time points followingdifferentiation to build cardiac development transcriptional landscapes.We performed RNA-seq experiments on day 0 to day 7 samples, as well asday 19 and day 35 samples (two independent biological replicates pertime point). Two batches of RNA-seq (100 bp and 50 bp read length) wereperformed using the illumine Hiseq 2000 platform. In total, 20 sampleswere examined. Bowtie and Tophat were used to map our reads into areference human genome (hg19) and we calculate each gene expression(annotation of the genes according to Refseq) using RPKM method (Readsper kilobase transcript per million reads). Differentiation of hPSCs tocardiomyocytes involves five major cell types: pluripotent stem cells(day 0), mesoderm progenitors (day 1 to day 2), cardiac mesoderm cells(day 3 to day 4), heart field progenitors (day 5, day 6 and day 7), andcardiomyocytes (day 10 after).

Molecular mRNA analysis of cardiac differentiation from hPSCs using theHVPG protocol revealed dynamic changes in gene expression, withdown-regulation of the pluripotency markers OCT4, NANOG and SOX2 duringdifferentiation. Induction of the primitive streak-like genes T andMIXL1 occurred within the first 24 hours following CHIR-98014 addition,and was followed by upregulation of the cardiac mesodermal marker MESP1on day 2 and day 3. Expression of the cardiac muscle markers TNNT2,TNNC1, MYL2, MYL7, MYH6, MYH7 and IRX4 was detected at later stage ofdifferentiation (after day 10).

By this analysis, genes enriched at each differentiation stage,including mesoderm cells, cardiac progenitors and cardiomyocytes, wereidentified. Mesoderm cells, which are related to day 1 differentiatedcells, express brachyury. We identified potential surface markers formesoderm cells, including: FZD10, CD48, CD1D, CD8B, IL15RA, TNFRSF1B,TNFSF13, ICOSLG, SEMA7A, SLC3A2, SDC1, HLA-A. Through similar analysis,we also identified surface markers for cardiac mesoderm mesp1 positivecells, including: CXCR4, ANPEP, ITGA5, TNFRSF9, FZD2, CD1D, CD177,ACVRL1, ICAM1, L1CAM, NGFR, ABCG2, FZD7, TNFRSF13C, TNFRSF1B.

Consistent with western blot analysis, ISL1 mRNA was expressed as earlyas day 4 and peaked on day 5, one day before its protein expressionreached its peak. On day 5 of differentiation (the cardiac progenitorstage, isl1 mRNA expression maximum on day 5, isl1 protein expressionmaximum on day 6), the day 5 enriched genes were compared with ananti-CD antibody array (a panel of 350 known CD antibodies) and a numberof potential cell-surface protein markers were identified. We identifiedmany cell-surface proteins expressed at this stage, including: FZD4,JAG1, PDGFRA, LIFR (CD118), TNFSF9, FGFR3.

The cell surface protein Jagged 1 (JAG1), Frizzled 4 (FZD4), LIFR(CD118) and FGFR3 were selected for further analysis. Jagged 1expression was further studied as described below and in Example 3.Frizzled 4 expression was further studied as described in Example 4.LIFR (CD118) and FGFR3 expression was further studied as described inExample 5.

Firstly, the expression of Isl1 and Jag1 was profiled using the doublestaining flow cytometry technique. Flow cytometric analysis was carriedout essentially as described in Example 1, using anti-Isl1 and anti-Jag1antibodies for double staining. Jagged 1 expression was found to tracethe expression of Islet 1 and on day 6 of differentiation, all of theIslet 1 positive cells also expressed Jagged 1, and vice versa. Becauseof the co-expression pattern of these two markers, a Jagged 1 antibodywas used to enrich the 94.1% Islet 1+ cells differentiated population to99.8% purity of Islet1+Jagged1+ cells.

It also was confirmed that Islet 1 is an earlier developmental gene thanthe Nkx2.5 gene using double immunostaining of ISL1 and NKX2.5expression in HVPs. The purified HVPs uniformly express the ISL1 gene,but at this stage, only a few of the cells started to express Nkx2.5.

Furthermore, immunostaining with both anti-Isl1 and anti-Jag 1 wasperformed, essentially as described in Example 1, on week 4 human fetalheart tissue, neonatal heart tissue and 8-year old heart tissue. Theresults revealed that in the in vivo fetal heart, all of the Islet 1positive cells also expressed Jagged 1. However, the neonatal heart and8-year old heart did not express Islet 1 or Jagged 1. In the ventricleof week 4 human fetal heart, cardiac Troponin T (cTnT) staining revealedvisible sarcomere structures. In addition, over 50% of ventricular cellsin the week 4 fetal heart expressed both Islet1 and Jagged1, which wasmarkedly decreased during subsequent maturation, with the loss ofexpression of both Islet1 and Jagged1 in the ventricular muscle cells ofthe human neonatal hearts.

The above-described experiments demonstrate that Jagged 1 is a cellsurface marker for Islet 1 positive cardiomyogenic progenitor cells.

Example 3: Clonal Differentiation of Isl1+Jag1+ Cardiac Progenitor Cells

To characterize the clonal differentiation potential of Isl1+Jag1+cells, cardiomyogenic progenitor cells were generated by the culturingprotocol described in Example 1, and one single Isl1+Jag1+ cell wasseeded into one well of a Matrigel-coated 48-well plate. Cells werepurified with antibody of Jag1 and then one single cell was seeded intoone well. The single cells were then cultured for 3 weeks in CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 2.5mM GlutaMAX, 100 μg/ml Vitamin C, 20% Knockout Serum Replacement).

Immunostaining of the 3-week differentiation cell population was thenperformed with three antibodies: cardiac troponin I (cTn1) forcardiomyocytes, CD144 (VE-cadherin) for endothelial cells and smoothmuscle actin (SMA) for smooth muscle cells. The results showed that thesingle cell-cultured, Isl1+Jag1+ cells gave rise to cTnI positive andSMA positive cells, but not VE-cadherin positive endothelial cells,indicating these generated Islet1+ cells are heart muscle progenitorsthat have limited differentiation potential to endothelial lineages.Purified Islet1+Jagged1+ cells differentiated with the HVPG protocolfrom human induced pluripotent stem cells (iPSC 19-9-11 line) alsoshowed similar in vitro differentiation potential and predominantlydifferentiate to cTnI+SMA+ cells, but not VE-cadherin+ cells. Over thecourse of several weeks, the cells expressed the ventricular specificmarker MLC2v, indicating that the initial ISL1+ subset was alreadycommitted to the ventricular cell fate. Because of the limited vasculardifferentiation potential of Islet1+ cells generated using the HVPGprotocol, these generated Islet1+ cells might represent a distinctprogenitor population from the previously reported KDR+ population(Yang, L. et al. (2008) Nature 453:524-528) or multipotent ISL1+ cells(Bu, L. et al. (2009) Nature 460:113-117; Moretti, A. et al. (2006) Cell127:1151-1165), which can give rise to all three lineages ofcardiovascular cells.

These results demonstrated that the Isl1+Jag1+ cardiomyogenic progenitorcells can be successfully cultured in vitro from a single cell to asignificantly expanded cell population (1×10⁹ cells or greater) thatcontains all three types of cardiac lineage cells, with a predominanceof cardiomyocytes. Furthermore, these cells can be cultured in vitro forextended periods of time, for at least 2-3 weeks, and even for months(e.g., six months or more). Since the cardiomyogenic progenitor cellsgradually differentiate into cardiomyocytes, which do not proliferate, aculture period of approximately 2-3 weeks is preferred.

Example 4: Identification of Frizzled 4 as a Cell Surface Marker ofCardiac Progenitor Cells

As described in Example 2, Frizzled 4 (FZD4) was identified by RNA-seqanalysis as being expressed in cardiac progenitor cells. Thus, toconfirm FZD4 as a cell surface marker of cardiac progenitor cells, FZD4expression was assessed during cardiac differentiation via Western blotanalysis. The results demonstrated that FZD4 was not expressed inpluripotent stem cells and the first 3 days differentiated cells.However, FZD4 started to express on day 4 and maximize its expression onday 5 of expression.

In order to quantify the co-expression pattern of FZD4 and Isl1 at thesingle cell level, FACS analysis was performed. On day 5 ofdifferentiation, more than 83% of cells express both isl1 and FZD4,demonstrating that FZD4 is a cell surface marker for Isl1 positive cellsduring cardiac progenitor differentiation using the GiWi protocol.

In order to confirm that both JAG1 and FZD4 were indeed co-expressedwith ISL1 on the human ventricular progenitor cells, tripleimmunofluorescence analysis of day 6 differentiated cells from hPSCs wasperformed with antibodies to Islet 1, Jagged 1 and Frizzled 4. Thetriple staining experiment demonstrated that Isl1+ cells expressed bothJagged 1 and Frizzled 4.

Example 5: Identification of Leukemia Inhibitor Factor Receptor (LIFR)and Fibroblast Growth Factor Receptor 3 (FGFR3) as Cell Surface Markersof Cardiac Progenitor Cells

As described in Example 2, LIFR(CD118) and FGFR3 were identified byRNA-seq analysis as being expressed in cardiac progenitor cells. In thisexample, expression of these additional cell surface markers for thehuman ventricular progenitor cells was confirmed by flow cytometryanalysis. Human ventricular progenitor (HVP) cells were generated asdescribed in Example 1 or 11 and day 6 cells were analyzed by standardflow cytometry. A double staining flow cytometry experiment usinganti-Islet 1 and anti-Leukemia Inhibitory Factor Receptor (LIFR)antibodies was performed. The results demonstrate that the HVP cellsco-express Islet 1 and LIFR, thereby confirming that LIFR is a cellsurface marker for the HVP cells. Furthermore, flow cytometryexperiments were performed comparing the expression of LIFR andFibroblast Growth Factor Receptor 3 (FGFR3) on day 6 HVP cells toundifferentiated embryonic stem (ES) cells. The results demonstrate thatLIFR and FGFR3 are both highly enriched for expression on the HVP cells,thereby confirming that LIFR and FGFR3 are both cell surface markers forthe HVP cells.

Example 6: In Vivo Developmental Potential of Isl1+Jag1+ CardiacProgenitor Cells'

The ES03 human embryonic stem cell (hESC) line (obtained from WiCellResearch Institute) expresses green fluorescent protein (GFP) driven bythe cardiac-specific cTnT promoter. ES03 cells were used to generateIsl1+Jag1+ cardiomyogenic progenitor cells using the culturing protocoldescribed in Example 1. The Isl1+Jag1+ cardiomyogenic progenitor cellswere transplanted into the hearts of severe combined immunodeficient(SCID) beige mice to document their developmental potential in vivo.

Briefly, Isl1+Jag1+ cells were injected (1,000,000 cells per recipient)directly into the left ventricular wall of NOD/SCID-gamma mice in anopen-chest procedure. Hearts were harvested 2-3 weeks post surgery,fixed in 1% PFA and sectioned at 10 μm (n=12). Histological analyses ofthe hearts of the transplanted mice revealed the presence of GFP+ donorcells, detected by epifluorescence and by staining with an anti-GFPantibody, demonstrating that the Isl1+Jag1+ cardiomyogenic progenitorcells were capable of differentiating into cardiomyocytes whentransplanted in vivo.

The Isl1+Jag1+ cardiomyogenic progenitor cells were also transplanteddirectly into infarcted hearts of SCID beige mice (“injured mice”), ascompared to similarly transplanted normal mice. When analyzed two weekslater, injured mice transplanted with the Isl1+Jag1+ cardiomyogenicprogenitor cells had a larger graft size than the normal mice similarlytransplanted, demonstrating the cardiomyocyte regeneration capacity ofthe Isl1+Jag1+ cardiomyogenic progenitor cells in vivo.

Example 7: Human Ventricular Progenitors (HPVs) Generate a 3-DVentricular Heart Muscle Organ In Vivo

The building of the ventricular heart muscle chamber is one of the mostcritical and earliest steps during human organogenesis, and requires aseries of coordinated steps, including migration, proliferation,vascularization, assembly, and matrix alignment. To test the capacity ofHVPs to drive ventriculogenesis in vivo, we transplanted purified HVPsor unpurified HVPs (92.0±1.9% ISL1+) under the kidney capsule ofimmunocompromised mice. After 2 months post-transplantation, animalstransplanted with unpurified HVPs formed tumors, resulting in a tumorformation efficiency of 100% (100%, 4/4), whereas animals transplantedwith purified HVPs did not form any tumors (0%, 0/10).

The engrafted kidneys with purified HVPs were further assayed forhistological analysis. Hematoxylin and Eosin (H&E) staining revealed anorgan that exceeded 0.5 cm in length with more than 1 mm thickness onthe surface of the mouse kidney, and that uniformly expressed theventricular specific marker MLC2v (O'Brien, T. X. et al. (1993) Proc.Natl. Acad. Sci. USA 90:5157-5161). The resulting human muscle organ wasfully vascularized and red blood cells could be detected in the bloodvessels. Analysis of cTnT, MLC2v, and MLC2a immunostaining furtherrevealed that the transplanted HVPs not only differentiated into cardiacmuscle cells (cTnT+ cells), but also further mature to become MLC2v+ventricular myocytes that are negative for MLC2a expression. Theresulting ventricular muscle organ is fully vascularized by murinederived vascular cells, consistent with the notion that itsvascularization occurred via paracrine cues derived from the HVPs.

The blood vessel structured was revealed by immunostaining analysis ofantibodies directed against VE-cadherin and smooth muscle actinexpression. In addition, using a human specific monoclonal lamininantibody targeting laminin γ-1 chain, the HVPs secreted their own humanlaminin as their extracellular matrix (the mouse kidney region isnegative for human laminin immunostaining). In addition, we found humanfibronectin expression is restricted to areas near the blood vesselsusing a monoclonal human fibronectin antibody.

To assess the capacity of late stage cardiac cells to driveventriculogenesis, NKX2.5+ cells (day 10 after differentiation) weretransplanted under the kidney capsule of immunocompromised NSG mice. Atthree weeks post-transplantation, animals transplanted with NKX2.5+cells did not form any visible human muscle graft, indicating that HVPslose their ability for in vivo ventriculogenesis following peak Islet-1expression.

Taken together, these studies indicate that the HVPs can synthesize andrelease their own cardiac laminin-derived matrix, as well as fibronectinwhich serves to stabilize the vasculature to the nascent ventricularorgan.

Example 8: HVPs Create a Mature, Functioning Ventricular Muscle Organ InVivo Via a Cell Autonomous Pathway

One of the critical limitations for the utility of hPSCs for studies ofhuman cardiac biology and disease is their lack of maturity andpersistence of expression of fetal isoforms. To determine if the HVPderived organs could become functional mature ventricular muscle, longterm transplantation studies were performed followed by detailedanalyses of a panel of well accepted features of adult ventricularmyocardium including formation of T tubules (Brette, F. and Orchard, C.(2003) Circ. Res. 92:1182-1192; Marks, A. R. (2013) J. Clin. Invest.123:46-52), ability to generate force comparable to other studies ofengineered ventricular tissue, loss of automaticity, and acquisition ofadult-rod shaped ventricular cardiomyocytes.

After 5 months post-transplantation of purified HVPs, no tumors formedin all of our animals. Animals were sacrificed and the engrafted kidneyswere removed for further analysis. The 5-month human graft was ahemisphere structure with the radius of 0.4 cm (diameter of 0.8 cm). Thevolume for the 5-month human graft was around 0.13 cm³ for one kidney, avolume that suggests feasibility for generating human ventricular musclethat achieves a thickness comparable to the in vivo human adult heart.Rod-shaped mature human ventricular myocytes were observed in the humanmuscle organ. In addition, muscle trips taken from our mature humanmuscle organ generated forces (0.36±0.04 mN) in response to electricstimulation and increased their force generation after treatment with aβ-adrenergic agonist isoprenaline (0.51±0.02 mN, p<0.05 compared tocontrol). Taken together, these studies indicate that the HVPs arecapable of generating a fully functional, mature human ventricularmuscle organ in vivo via a cell autonomous pathway, i.e., without theaddition of other cells, genes, matrix proteins, or biomaterials.

Example 9: HVPs Migrate Towards an Epicardial Niche and SpontaneouslyForm a Human Ventricular Muscle Patch on the Surface of a Normal MurineHeart In Vivo

The epicardium is a known niche for heart progenitors, driving thegrowth of the ventricular chamber during compact zone expansion, as wellas serving as a home to adult epicardial progenitors that can expandafter myocardial injury and that can drive vasculogenesis in response toknown vascular cell fate switches, such as VEGF (Giordano, F. J. et al.(2001) Proc. Natl. Acad. Sci. USA 98:5780-5785; Masters, M. and Riley,P. R. (2014) Stem Cell Res. 13:683-692; Zangi, L. et al. (2013) Nat.Biotechnol. 31:898-907). To determine if the HVPs might migratespontaneously to the epicardial surface of the normal heart, purifiedgreen fluorescent protein (GFP)-labeled HVPs were injectedintra-myocardially into the hearts of immunocompromised mice. After oneweek or one month post-transplantation, animals were sacrificed and theengrafted hearts were removed for histology. After one weekpost-transplantation, the majority of GFP+ cells were retained in themyocardium. However, almost all the GFP+ cells migrated to theepicardium after one month post-transplantation. In addition, GFP+ cellswere ISL1+ and Ki67+ after one week post-transplantation.

In order to trace the differentiation potential of Islet1+ cells, thepurified ISL1+ JAG1+ cells generated from a cTnT promoter driven greenfluorescent protein (GFP)-expressing hESC line (H9-cTnT-GFP) weretransplanted into the hearts of severe combined immunodeficient (SCID)beige mice to document their developmental potential in vivo. One monthafter transplantation of Isl1+Jag1+ cells directly into the ventricle ofthe hearts of SCID beige mice, Hematoxylin and eosin staining revealed ahuman muscle strip graft present in the epicardium of the murine heart.In addition, immunohistological analyses revealed the presence of GFP+donor cells detected by epifluorescence and by staining with an anti-GFPantibody. More importantly, when analysed with antibodies of MLC2v andMLC2a, the grafted human muscle strip is positive for MLC2v (100% ofcells +), and negative for the atrial marker MLC2a, indicating thetransplanted ISL1+ cells not only further differentiated to cardiacmuscle cells, but also became ventricular muscle cells.

Taken together, these studies indicate that the HVPs can migrate to anepicarial niche, where they expand, and subsequently differentiate in toa homogenous ventricular muscle patch, again without the addition ofexogenous cells, genes, matrices, or biomaterials.

Example 10: Additional Experimental Materials and Methods

In this example, additional details on the experimental materials andmethods used in Examples 1-9 are provided.

Maintenance of hPSCs

hESCs (ES03, H9) and human iPSCs (19-9-11) were maintained on Matrigel(BD Biosciences) coated plates in mTeSR1 medium (STEMCELL Technologies)according to previous published methods (Lian, X. et al. (2013) Nat.Proc. 8:162-175; Lian, X. et al. (2013) Stem Cells 31:447-457).

Human Ventricular Progenitor Generation (HVPG) Protocol

hPSCs maintained on a Matrigel-coated surface in mTeSR1 were dissociatedinto single cells with Accutase at 37° C. for 10 min and then seededonto a Matrigel-coated cell culture dish at 100,000-200,000 cell/cm² inmTeSR1 supplemented with 5 μM ROCK inhibitor Y-27632 (day −2) for 24hours. At day −1, cells were cultured in mTeSR1. At day 0, cells weretreated with 1 μM CHIR-98014 (Selleckchem) in RPMI supplemented with B27minus insulin (RPMI/B27-ins) for 24 hours (day 0 to day 1), which wasthen removed during the medium change on day 1. At day 3, half of themedium was changed to the RPMI/B27-ins medium containing 2 μM Wnt-059(Selleckchem), which was then removed during the medium change on day 5.At day 6, cells were dissociated into single cells and purified withanti-JAG1 or anti-FZD4 antibody.

RNA-Seq Library Construction

RNA was isolated (RNeasy Mini kit, Qiagen), quantified (Qubit RNA AssayKit, Life Technologies) and quality controlled (BioAnalyzer 2100,Agilent). RNA (800 ng) from each sample was used as input for theIllumina TruSeq mRNA Sample Prep Kit v2 (Illumina) and sequencinglibraries were created according to the manufacturer's protocol.Briefly, poly-A containing mRNA molecules were purified using poly-Toligo-attached magnetic beads. Following purification, the mRNA wasfragmented and copied into first strand complementary DNA using randomprimers and reverse transcriptase. Second strand cDNA synthesis was thendone using DNA polymerase I and RNase H. The cDNA was ligated toadapters and enriched with PCR to create the final cDNA library. Thelibrary was pooled and sequenced on a HiSeq 2000 (Illumina) instrumentper the manufacturer's instructions.

RNA-Seq Data Processing

The RNA-seq reads were trimmed and mapped to the hg19 reference usingTophat 2. On average, approximately 23 million reads were generated persample, and 76% of these reads were uniquely mapped. Expression levelsfor each gene were quantified using the python script rpkmforgenes andannotated using RefSeq. Genes without at least one sample with at leastten reads were removed from the analysis. Principle Component Analysisand heatmaps were constructed using the R and Gene-E respectively.

Transplantation

Aliquots of 2 million purified HVPs were collected into an eppendorftube. Cells were spun down, and the supernatant was discarded. Each tubeof cells was transplanted under the kidney capsule, orintra-myocardially injected into the heart of the immunodeficient mice,NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ or SCID-Beige respectively (CharlesRiver France), following a previously described protocol (Shultz, L. D.et al. (2005) J. Immunol. 174:6477-6489). Engrafted Kidneys or heartsare harvested at various time intervals for histological andphysiological analysis.

Flow Cytometry

Cells were dissociated into single cells with Accutase for 10 min andthen fixed with 1% paraformaldehyde for 20 min at room temperature andstained with primary and secondary antibodies in PBS plus 0.1% TritonX-100 and 0.5% BSA. Data were collected on a FACSCaliber flow cytometer(Beckton Dickinson) and analyzed using FlowJo.

Immunostaining

Cells were fixed with 4% paraformaldehyde for 15 min at room temperatureand then stained with primary and secondary antibodies in PBS plus 0.4%Triton X-100 and 5% non-fat dry milk (Bio-Rad). Nuclei were stained withGold Anti-fade Reagent with DAPI (Invitrogen). An epifluorescencemicroscope and a confocal microscope (ZEISS, LSM 700) were used forimaging analysis.

Western Blot Analysis

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce)in the presence of Halt Protease and Phosphatase Inhibitor Cocktail(Pierce). Proteins were separated by 10% Tris-Glycine SDS/PAGE(Invitrogen) under denaturing conditions and transferred to anitrocellulose membrane. After blocking with 5% dried milk in TBST, themembrane was incubated with primary antibody overnight at 4° C. Themembrane was then washed, incubated with an anti-mouse/rabbitperoxidase-conjugated secondary antibody at room temperature for 1 hour,and developed by SuperSignal chemiluminescence (Pierce).

Electrophysiology (Patch Clamping)

Beating ventricular myocyte clusters were microdissected and replatedonto glass coverslips before recording. Action potential activity wasassessed using borosilicate glass pipettes (4-5 M Ohm resistance) filledwith intracellular solution consisting of 120 mM K D-gluconate, 25 mMKCl, 4 mM MgATP, 2 mM NaGTP, 4 mM Na2-phospho-creatin, 10 mM EGTA, 1 mMCaCl2, and 10 mM HEPES (pH 7.4 adjusted with HCl at 25° C.). Culturedcardiomyocytes seeded on coverslip dishes were submerged inextracellular solution (Tyrode's solution) containing 140 mM NaCl, 5.4mM KCl, 1 mM MgCl2, 10 mM glucose, 1.8 mM CaCl2, and 10 mM HEPES (pH 7.4adjusted with NaOH at 25° C.). Spontaneous action potentials wererecorded at 37° C. using patch clamp technique (whole-cell, currentclamp configuration) performed using a Multiclamp 700B amplifier(Molecular Devices, CA, USA) software low-pass filtered at 1 kHz,digitized and stored using a Digidata 1322A and Clampex 9.6 software(Molecular Devices, CA, USA).

Statistics

Data are presented as mean±standard error of the mean (SEM). Statisticalsignificance was determined by Student's t-test (two-tail) between twogroups. P<0.05 was considered statistically significant.

Example 11: Xeno-Free Human Ventricular Progenitor DifferentiationProtocol

In this example, an alternative differentiation protocol fordifferentiation of human ventricular progenitors is provided, whichutilizes a defined, xeno-free culture medium, Essential 8. The Essential8 medium was developed for growth and expansion of human pluripotentstem cells (hPSCs) and is described further in Chen, G. et al. (2011)Nat. Methods 8:424-429 (referred to therein as “E8” medium).

hPSCs maintained on a Vitronectin (or Laminin 521)-coated surface inEssential 8 medium were dissociated into single cells with Versenesolution at 37° C. for 10 min and then seeded onto a Vitronectin (orLaminin 521)-coated cell culture dish at 100,000-200,000 cell/cm² inEssential 8 medium supplemented with 5 μM ROCK inhibitor Y-27632 (day−2) for 24 hours. At day −1, cells were cultured in Essential 8 medium.At day 0, cells were treated with 0.5 μM CHIR-98014 in RPMI for 24 hours(day 0 to day 1), which was then removed during the medium change onday 1. At day 3, half of the medium was changed to the RPMI mediumcontaining 0.5 μM Wnt-059, which was then removed during the mediumchange on day 5. At day 6, cells (human ventricular progenitors) weredissociated into single cells and purified with anti-JAG1 or anti-FZD4antibody. Alternatively cells are purified with anti-LIFR, anti-FGFR3antibody or anti-TNFSF9 antibody.

Example 12: Angiogenic Markers for Engraftable Human VentricularProgenitor Cells

In this example, genes in the angiogenic family that are expressed inhuman ventricular progenitor cells (HVPs) were identified. HVPs weregenerated as described in Examples 1 or 11 and RNA sequencing (RNA-seq)was performed at different time points following differentiation asdescribed in Example 2. Cluster analysis of gene expression profiles atdifferent time points during HVP differentiation identifiedstage-specific signature genes. These genes were clusteredhierarchically on the basis of the similarity of their expressionprofiles. First, genes showing expression in four different categorieswere identified: (i) cell surface expression; (ii) co-expression withIslet 1; (iii) high expression on day 5 of differentiation; and (iv)high d5/d0 ratio. This analysis confirmed the cell surface markers forHVPs of: JAG1, FZD4, FGFR3, LIFR (CD118) and TNFSF9. Next, from thissame population of HVPs that identified the cell surface markers, geneontogeny searches were performed to identify angiogenic family genesthat were expressed in this population of HVPs, to thereby identify agene fingerprint profile that identifies genes critical for cellengraftment.

Statistically, Pearson's correlation with Isl1 expression was used toidentify those angiogenic genes whose expression in the HVPs bestcorrelated with Isl1 expression. Table 1 below lists the angiogenicgenes that correlate with Isl1 expression with a Pearson's correlationof 0.50 or higher.

TABLE 1 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of 0.50 or greater Pearson's Correlation with Isl1Gene Angiogenic genes (GO: 0001525) Expression FGF10 fibroblast growthfactor 10 0.98 PRKD1 protein kinase D1 0.95 CCBE1 collagen and calciumbinding EGF domains 1 0.94 PDGFRA platelet-derived growth factorreceptor, 0.94 alpha polypeptide EPHB2 EPH receptor B2 0.92 GATA2 GATAbinding protein 2 0.92 NTRK1 neurotrophic tyrosine kinase, receptor,type 1 0.92 PTGIS prostaglandin I2 (prostacyclin) synthase 0.87 BMPERBMP binding endothelial regulator 0.85 BMP4 bone morphogenetic protein 40.84 C1GALT1 core 1 synthase, glycoprotein-N-acetyl- 0.84 galactosamine3-beta-galactosyltransferase 1 MEIS1 Meis homeobox 1 0.83 TBX1 T-box 10.83 PKNOX1 PBX/knotted 1 homeobox 1 0.83 ID1 inhibitor of DNA binding1, dominant 0.82 negative helix-loop-helix protein TCF21 transcriptionfactor 21 0.82 HEY1 hes-related family bHLH transcription 0.80 factorwith YRPW motif 1 HOXB3 homeobox B3 0.78 JAG1 jagged 1 0.75 HGFhepatocyte growth factor (hepapoietin A; 0.74 scatter factor) IL6interleukin 6 0.74 GHRL ghrelin/obestatin prepropeptide 0.73 IHH indianhedgehog 0.70 SRPK2 SRSF protein kinase 2 0.70 GATA6 GATA bindingprotein 6 0.69 HAND1 heart and neural crest derivatives expressed 1 0.69AMOT angiomotin 0.69 NRP2 neuropilin 2 0.65 PTEN phosphatase and tensinhomolog 0.65 SEMA3E sema domain, immunoglobulin domain 0.64 (Ig), shortbasic domain, secreted, (semaphorin) 3E APOLD1 apolipoprotein L domaincontaining 1 0.62 SETD2 SET domain containing 2 0.62 DAB2IP DAB2interacting protein 0.61 KDR kinase insert domain receptor 0.60 PGFplacental growth factor 0.60 EMP2 epithelial membrane protein 2 0.59TAL1 T-cell acute lymphocytic leukemia 1 0.58 ACVR1 activin A receptor,type I 0.58 HIPK2 homeodomain interacting protein kinase 2 0.56 CSPG4chondroitin sulfate proteoglycan 4 0.55 TNFAIP3 tumor necrosis factor,alpha-induced 0.55 protein 3 NRP1 neuropilin 1 0.55 NFATC4 nuclearfactor of activated T-cells, 0.54 cytoplasmic, calcineurin-dependent 4CDC42 cell division cycle 42 0.54 ANGPTL4 angiopoietin-like 4 0.53 BCAS3breast carcinoma amplified sequence 3 0.53 HIPK1 homeodomain interactingprotein kinase 1 0.53 NRXN3 neurexin 3 0.52 FZD5 frizzled class receptor5 0.52 HHEX hematopoietically expressed homeobox 0.50

Table 2 below lists the angiogenic genes that correlate with Isl1expression with a Pearson's correlation of 0.49-0.00.

TABLE 2 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of 0.49 to 0.00 Pearson's Correlation with Isl1Gene Angiogenic genes (GO: 0001525) Expression ACVRL1 activin A receptortype II-like 1 0.49 ENPEP glutamyl aminopeptidase (aminopeptidase A)0.49 EFNA1 ephrin-A1 0.49 CHRNA7 cholinergic receptor, nicotinic, alpha7 0.49 (neuronal) TMEM100 transmembrane protein 100 0.48 NOS3 nitricoxide synthase 3 (endothelial cell) 0.47 LEF1 lymphoid enhancer-bindingfactor 1 0.47 NRXN1 neurexin 1 0.46 EPHB3 EPH receptor B3 0.44 ROCK1Rho-associated, coiled-coil containing 0.42 protein kinase 1 NF1neurofibromin 1 0.42 CYSLTR2 cysteinyl leukotriene receptor 2 0.42 FGFR2fibroblast growth factor receptor 2 0.41 GATA4 GATA binding protein 40.40 FMNL3 formin-like 3 0.40 C3 complement component 3 0.40 WASF2 WASprotein family, member 2 0.40 CALCRL calcitonin receptor-like 0.39 HIF1Ahypoxia inducible factor 1, alpha subunit 0.39 (basic helix-loop- helixtranscription factor) VEGFA vascular endothelial growth factor A 0.39KRIT1 KRIT1, ankyrin repeat containing 0.39 CDH13 cadherin 13 0.39COL18A1 collagen, type XVIII, alpha 1 0.39 STK4 serine/threonine kinase4 0.38 C5 complement component 5 0.38 HDAC7 histone deacetylase 7 0.38ANGPT2 angiopoietin 2 0.38 PLCG1 phospholipase C, gamma 1 0.37 EDNRAendothelin receptor type A 0.35 TGFB2 transforming growth factor, beta 20.35 HAND2 heart and neural crest derivatives expressed 2 0.35 CD34 CD34molecule 0.35 BTG1 B-cell translocation gene 1, anti-proliferative 0.34TGFBR1 transforming growth factor, beta receptor 1 0.33 FGFR1 fibroblastgrowth factor receptor 1 0.33 FN1 fibronectin 1 0.31 TWIST1 twist familybHLH transcription factor 1 0.31 ELK3 ELK3, ETS-domain protein (SRFaccessory 0.30 protein 2) THSD7A thrombospondin, type I, domaincontaining 0.30 7A RGCC regulator of cell cycle 0.30 PLCD1 phospholipaseC, delta 1 0.29 SPARC secreted protein, acidic, cysteine-rich 0.29(osteonectin) TBX20 T-box 20 0.28 PIK3CAphosphatidylinositol-4,5-bisphosphate 0.27 3-kinase, catalytic subunitalpha MMRN2 multimerin 2 0.27 FOXO4 forkhead box O4 0.26 RAMP2 receptor(G protein-coupled) activity 0.25 modifying protein 2 FLT1 fms-relatedtyrosine kinase 1 0.25 ADRB2 adrenoceptor beta 2, surface 0.25 SLC12A6solute carrier family 12 (potassium/chloride 0.25 transporter), member 6ADM adrenomedullin 0.25 NPPB natriuretic peptide B 0.24 SPINK5 serinepeptidase inhibitor, Kazal type 5 0.24 MAPK14 mitogen-activated proteinkinase 14 0.24 MMP2 matrix metallopeptidase 2 0.24 PTPRM proteintyrosine phosphatase, receptor 0.23 type, M OVOL2 ovo-like zinc finger 20.23 CTNNB1 catenin (cadherin-associated protein), beta 1, 0.22 88 kDaOTULIN OTU deubiquitinase with linear linkage 0.21 specificity B4GALT1UDP-Gal:betaGlcNAc beta 1,4-galactosyl- 0.21 transferase, polypeptide 1PDGFRB platelet-derived growth factor receptor, beta 0.20 polypeptide F3coagulation factor III (thromboplastin, tissue 0.20 factor) PRKCAprotein kinase C, alpha 0.20 LRP5 low density lipoproteinreceptor-related 0.20 protein 5 MAP3K7 mitogen-activated protein kinasekinase 0.20 kinase 7 NRCAM neuronal cell adhesion molecule 0.19 MAP2K5mitogen-activated protein kinase kinase 5 0.18 S1PR1sphingosine-1-phosphate receptor 1 0.18 NFATC3 nuclear factor ofactivated T-cells, 0.18 cytoplasmic, calcineurin-dependent 3 TSPAN12tetraspanin 12 0.18 LAMA5 laminin, alpha 5 0.17 LOXL2 lysyl oxidase-like2 0.17 ANGPT1 angiopoietin 1 0.17 GTF2I general transcription factor IIi0.16 E2F8 E2F transcription factor 8 0.16 PDE3B phosphodiesterase 3B,cGMP-inhibited 0.15 SHB Src homology 2 domain containing adaptor 0.14protein B MYH9 myosin, heavy chain 9, non-muscle 0.14 FZD8 frizzledclass receptor 8 0.14 NOV nephroblastoma overexpressed 0.14 SH2D2A SH2domain containing 2A 0.14 FGF8 fibroblast growth factor 8(androgen-induced) 0.13 TIE1 tyrosine kinase with immunoglobulin-like0.13 and EGF-like domains 1 EGLN1 egl-9 family hypoxia-inducible factor1 0.12 RORA RAR-related orphan receptor A 0.11 MFGE8 milk fatglobule-EGF factor 8 protein 0.11 ARHGAP24 Rho GTPase activating protein24 0.10 ITGA5 integrin, alpha 5 (fibronectin receptor, alpha 0.10polypeptide) PARVA parvin, alpha 0.10 ADIPOR2 adiponectin receptor 20.09 NPR1 natriuretic peptide receptor 1 0.09 ITGB1 integrin, beta 1(fibronectin receptor, beta 0.09 polypeptide, antigen CD29 includesMDF2, MSK12) HIF3A hypoxia inducible factor 3, alpha subunit 0.08 EPAS1endothelial PAS domain protein 1 0.08 FOXC2 forkhead box C2 0.07 ANXA2annexin A2 0.06 RBM15 RNA binding motif protein 15 0.06 PITX2paired-like homeodomain 2 0.06 FOXC1 forkhead box C1 0.06 SRF serumresponse factor 0.06 ECSCR endothelial cell surface expressed chemotaxis0.05 and apoptosis regulator SOX17 SRY (sex determining region Y)-box 170.04 HDAC5 histone deacetylase 5 0.04 LRG1 leucine-richalpha-2-glycoprotein 1 0.04 ADAM8 ADAM metallopeptidase domain 8 0.03UBP1 upstream binding protein 1 (LBP-1a) 0.02 VASH1 vasohibin 1 0.02ANXA3 annexin A3 0.01 RRAS related RAS viral (r-ras) oncogene homolog0.01 TYMP thymidine phosphorylase 0.01 PRCP prolylcarboxypeptidase(angiotensinase C) 0.01 SEMA5A sema domain, seven thrombospondin repeats0.00 (type 1 and type 1-like), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 5A GREM1 gremlin 1, DAN family BMPantagonist 0.00

Angiogenic genes whose expression negatively correlated with Isl1expression in the HVPs were also identified. Table 3 below lists theangiogenic genes that negatively correlate with Isl1 expression with aPearson's correlation of −0.50 or less.

TABLE 3 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of −0.50 or less Pearson's Correlation with Isl1Gene Angiogenic genes (GO: 0001525) Expression ETS1 v-ets avianerythroblastosis virus E26 −0.50 oncogene homolog 1 BAX BCL2-associatedX protein −0.50 XBP1 X-box binding protein 1 −0.52 TDGF1teratocarcinoma-derived growth factor 1 −0.53 C5AR1 complement component5a receptor 1 −0.53 EPHA1 EPH receptor A1 −0.53 HS6ST1 heparan sulfate6-O-sulfotransferase 1 −0.56 SHC1 SHC (Src homology 2 domain containing)−0.56 transforming protein 1 SP100 SP100 nuclear antigen −0.58 JAM3junctional adhesion molecule 3 −0.58 CASP8 caspase 8, apoptosis-relatedcysteine −0.60 peptidase FLT4 fms-related tyrosine kinase 4 −0.60 SFRP2secreted frizzled-related protein 2 −0.61 HPSE heparanase −0.61 BAK1BCL2-antagonist/killer 1 −0.65 GPX1 glutathione peroxidase 1 −0.65 VAV3vav 3 guanine nucleotide exchange factor −0.70 VAV2 vav 2 guaninenucleotide exchange factor −0.72 EGF epidermal growth factor −0.72ADAM15 ADAM metallopeptidase domain 15 −0.73 AGGF1 angiogenic factorwith G patch and FHA −0.76 domains 1Table 4 below lists the angiogenic genes that negatively correlate withIsl1 expression with a Pearson's correlation of −0.01 to −0.49

TABLE 4 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of −0.01 to −0.49 Pearson's Correlation with Isl1Gene Angiogenic genes (GO: 0001525) Expression EIF2AK3 eukaryotictranslation initiation factor 2- −0.01 alpha kinase 3 ROCK2Rho-associated, coiled-coil containing −0.01 protein kinase 2 WNT5Awingless-type MMTV integration site −0.02 family, member 5A NR4A1nuclear receptor subfamily 4, group A, −0.02 member 1 CYP1B1 cytochromeP450, family 1, subfamily B, −0.02 polypeptide 1 PTK2 protein tyrosinekinase 2 −0.03 SFRP1 secreted frizzled-related protein 1 −0.04 STAT1signal transducer and activator of −0.04 transcription 1, 91 kDa ITGAVintegrin, alpha V −0.04 EPHB4 EPH receptor B4 −0.05 CYR61 cysteine-rich,angiogenic inducer, 61 −0.05 TEK TEK tyrosine kinase, endothelial −0.06COL15A1 collagen, type XV, alpha 1 −0.06 COL4A1 collagen, type IV, alpha1 −0.07 ANG angiogenin, ribonuclease, RNase A family, 5 −0.07 HSPB1 heatshock 27 kDa protein 1 −0.07 PLXND1 plexin D1 −0.08 HSPG2 heparansulfate proteoglycan 2 −0.09 VEGFC vascular endothelial growth factor C−0.09 SYNJ2BP synaptojanin 2 binding protein −0.09 THBS1 thrombospondin1 −0.09 CTGF connective tissue growth factor −0.10 ITGB3 integrin, beta3 (platelet glycoprotein IIIa, −0.12 antigen CD61) AAMPangio-associated, migratory cell protein −0.12 GJA5 gap junctionprotein, alpha 5, 40 kDa −0.12 PRKCB protein kinase C, beta −0.13 EGR3early growth response 3 −0.13 JMJD6 jumonji domain containing 6 −0.13TGFBI transforming growth factor, beta-induced, −0.14 68 kDa SIRT1sirtuin 1 −0.14 ANGPTL3 angiopoietin-like 3 −0.14 ACKR3 atypicalchemokine receptor 3 −0.14 SAT1 spermidine/spermine N1-acetyltransferase1 −0.15 VEGFB vascular endothelial growth factor B −0.16 UTS2 urotensin2 −0.16 JUN jun proto-oncogene −0.16 TNFSF12 tumor necrosis factor(ligand) superfamily, −0.16 member 12 EGFL7 EGF-like-domain, multiple 7−0.17 MED1 mediator complex subunit 1 −0.17 SLIT2 slit guidance ligand 2−0.17 SERPINF1 serpin peptidase inhibitor, clade F (alpha-2 −0.18antiplasmin, pigment epithelium derived factor), member 1 NOTCH3 notch 3−0.18 FGF9 fibroblast growth factor 9 −0.19 DLL4 delta-like 4(Drosophila) −0.19 CCL2 chemokine (C-C motif) ligand 2 −0.19 MMP14matrix metallopeptidase 14 (membrane- −0.19 inserted) TMPRSS6transmembrane protease, serine 6 −0.19 EPGN epithelial mitogen −0.20RBPJ recombination signal binding protein for −0.20 immunoglobulin kappaJ region COL4A2 collagen, type IV, alpha 2 −0.20 PRKD2 protein kinase D2−0.20 ALOX12 arachidonate 12-lipoxygenase −0.21 RNH1ribonuclease/angiogenin inhibitor 1 −0.21 APOH apolipoprotein H(beta-2-glycoprotein I) −0.21 CHI3L1 chitinase 3-like 1 (cartilageglycoprotein-39) −0.21 ESM1 endothelial cell-specific molecule 1 −0.22PTGS2 prostaglandin-endoperoxide synthase 2 −0.22 (prostaglandin G/Hsynthase and cyclooxygenase) ANPEP alanyl (membrane) aminopeptidase−0.22 LEMD3 LEM domain containing 3 −0.22 UTS2R urotensin 2 receptor−0.22 CIB1 calcium and integrin binding 1 (calmyrin) −0.22 ITGB1BP1integrin beta 1 binding protein 1 −0.22 AQP1 aquaporin 1 (Colton bloodgroup) −0.22 IL18 interleukin 18 −0.22 EPHA2 EPH receptor A2 −0.22 EPHB1EPH receptor B1 −0.22 AGT angiotensinogen (serpin peptidase inhibitor,−0.22 clade A, member 8) PLAU plasminogen activator, urokinase −0.22VEZF1 vascular endothelial zinc finger 1 −0.23 SPHK1 sphingosine kinase1 −0.23 SRPX2 sushi-repeat containing protein, X-linked 2 −0.23 PDCL3phosducin-like 3 −0.23 COL8A1 collagen, type VIII, alpha 1 −0.24 HDAC9histone deacetylase 9 −0.24 CTSH cathepsin H −0.24 EDN1 endothelin 1−0.24 CXCL8 chemokine (C-X-C motif) ligand 8 −0.24 ECM1 extracellularmatrix protein 1 −0.24 BRCA1 breast cancer 1, early onset −0.24 EFNB2ephrin-B2 −0.25 SERPINE1 serpin peptidase inhibitor, clade E (nexin,−0.25 plasminogen activator inhibitor type 1), member 1 SASH1 SAM andSH3 domain containing 1 −0.25 WNT7B wingless-type MMTV integration site−0.25 family, member 7B RAMP1 receptor (G protein-coupled) activity−0.26 modifying protein 1 SCG2 secretogranin II −0.26 COL8A2 collagen,type VIII, alpha 2 −0.26 SULF1 sulfatase 1 −0.26 CLIC4 chlorideintracellular channel 4 −0.26 FGF1 fibroblast growth factor 1 (acidic)−0.27 NODAL nodal growth differentiation factor −0.27 RASIP1 Rasinteracting protein 1 −0.28 RLN2 relaxin 2 −0.28 POFUT1 proteinO-fucosyltransferase 1 −0.28 FGF18 fibroblast growth factor 18 −0.28AIMP1 aminoacyl tRNA synthetase complex- −0.28 interactingmultifunctional protein 1 TGFBR2 transforming growth factor, betareceptor II −0.28 (70/80 kDa) RHOB ras homolog family member B −0.28GBX2 gastrulation brain homeobox 2 −0.28 ENPP2 ectonucleotidepyrophosphatase/ −0.29 phosphodiesterase 2 MAPK7 mitogen-activatedprotein kinase 7 −0.30 PROK2 prokineticin 2 −0.30 E2F7 E2F transcriptionfactor 7 −0.30 ERAP1 endoplasmic reticulum aminopeptidase 1 −0.31 MTDHmetadherin −0.31 KLF5 Kruppel-like factor 5 (intestinal) −0.31 DICER1dicer 1, ribonuclease type III −0.32 LECT1 leukocyte cell derivedchemotaxin 1 −0.32 CX3CL1 chemokine (C-X3-C motif) ligand 1 −0.32 PTK2Bprotein tyrosine kinase 2 beta −0.33 SEMA4A sema domain, immunoglobulindomain (Ig), −0.34 transmembrane domain (TM) and short cytoplasmicdomain, (semaphorin) 4A ARHGAP22 Rho GTPase activating protein 22 −0.34RSPO3 R-spondin 3 −0.34 KLF4 Kruppel-like factor 4 (gut) −0.34 ROBO1roundabout guidance receptor 1 −0.34 GPLD1 glycosylphosphatidylinositolspecific −0.35 phospholipase D1 NUS1 NUS1 dehydrodolichyl diphosphate−0.35 synthase subunit NRARP NOTCH-regulated ankyrin repeat protein−0.35 PDCD10 programmed cell death 10 −0.36 PF4 platelet factor 4 −0.36PRKX protein kinase, X-linked −0.36 PML promyelocytic leukemia −0.36ATP5B ATP synthase, H+ transporting, mito- −0.36 chondrial F1 complex,beta polypeptide TNFRSF12A tumor necrosis factor receptor superfamily,−0.36 member 12A ENG endoglin −0.37 THY1 Thy-1 cell surface antigen−0.37 FGF2 fibroblast growth factor 2 (basic) −0.37 CXCL12 chemokine(C-X-C motif) ligand 12 −0.37 CAV1 caveolin 1, caveolae protein, 22 kDa−0.38 PDGFA platelet-derived growth factor alpha −0.38 polypeptidePNPLA6 patatin-like phospholipase domain −0.38 containing 6 PLCD3phospholipase C, delta 3 −0.38 DDAH1 dimethylargininedimethylaminohydrolase 1 −0.39 GNA13 guanine nucleotide binding protein−0.39 (G protein), alpha 13 ADM2 adrenomedullin 2 −0.39 HMOX1 hemeoxygenase 1 −0.40 MCAM melanoma cell adhesion molecule −0.41 RAPGEF3 Rapguanine nucleotide exchange factor −0.41 (GEF) 3 TNFAIP2 tumor necrosisfactor, alpha-induced −0.41 protein 2 HTATIP2 HIV-1 Tat interactiveprotein 2, 30 kDa −0.42 NCL nucleolin −0.42 ERBB2 erb-b2 receptortyrosine kinase 2 −0.43 NAA15 N(alpha)-acetyltransferase 15, NatA −0.43auxiliary subunit ATPIF1 ATPase inhibitory factor 1 −0.43 THBS4thrombospondin 4 −0.43 SYK spleen tyrosine kinase −0.44 LIF leukemiainhibitory factor −0.44 THBS2 thrombospondin 2 −0.44 PPP1R16B proteinphosphatase 1, regulatory −0.44 subunit 16B NOTCH1 notch 1 −0.44 RUNX1runt-related transcription factor 1 −0.45 PDCD6 programmed cell death 6−0.45 VASH2 vasohibin 2 −0.45 GPI glucose-6-phosphate isomerase −0.46ZC3H12A zinc finger CCCH-type containing 12A −0.46 WARStryptophanyl-tRNA synthetase −0.46 HYAL1 hyaluronoglucosaminidase 1−0.47 PIK3CB phosphatidylinositol-4,5-bisphosphate −0.47 3-kinase,catalytic subunit beta TNMD tenomodulin −0.49

Example 13: Extracellular Matrix Markers for Engraftable HumanVentricular Progenitor Cells

In this example, genes in the extracellular matrix family that areexpressed in human ventricular progenitor cells (HVPs) were identified.HVPs were generated as described in Examples 1 or 11 and RNA sequencing(RNA-seq) was performed at different time points followingdifferentiation as described in Example 2. Cluster analysis of geneexpression profiles at different time points during HVP differentiationidentified stage-specific signature genes. These genes were clusteredhierarchically on the basis of the similarity of their expressionprofiles. First, genes showing expression in four different categorieswere identified: (i) cell surface expression; (ii) co-expression withIslet 1; (iii) high expression on day 5 of differentiation; and (iv)high d5/d0 ratio. This analysis confirmed the cell surface markers forHVPs of: JAG1, FZD4, FGFR3, LIFR (CD118) and TNFSF9. Next, from thissame population of HVPs that identified the cell surface markers, geneontogeny searches were performed to identify extracellular matrix familygenes that were expressed in this population of HVPs, to therebyidentify a gene fingerprint profile that identifies genes critical forcell engraftment.

Statistically, Pearson's correlation with Isl1 expression was used toidentify those extracellular matrix genes whose expression in the HVPsbest correlated with Isl1 expression. Table 5 below lists theextracellular matrix genes that correlate with Isl1 expression with aPearson's correlation of 0.50 or higher.

TABLE 5 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of 0.50 or greater Pearson'sCorrelation with Isl1 Gene Extracellular matrix genes (GO: 0031012)Expression FGF10 fibroblast growth factor 10 0.98 SMOC1 SPARC relatedmodular calcium binding 1 0.97 CCBE1 collagen and calcium binding EGFdomains 1 0.94 COL6A6 collagen, type VI, alpha 6 0.89 ADAMTS12 ADAMmetallopeptidase with thrombospondin 0.85 type 1 motif, 12 COL19A1collagen, type XIX, alpha 1 0.85 LAMA1 laminin, alpha 1 0.85 BMP4 bonemorphogenetic protein 4 0.84 FBLN7 fibulin 7 0.81 FBLN2 fibulin 2 0.81NDNF neuron-derived neurotrophic factor 0.80 HTRA1 HtrA serine peptidase1 0.80 HAPLN1 hyaluronan and proteoglycan link protein 1 0.79 EMILIN1elastin microfibril interfacer 1 0.79 SPOCK3 sparc/osteonectin, cwcv andkazal-like domains 0.76 proteoglycan (testican) 3 PODNL1 podocan-like 10.73 IHH indian hedgehog 0.70 ACAN aggrecan 0.69 NID2 nidogen 2(osteonidogen) 0.69 COL4A6 collagen, type IV, alpha 6 0.68 LAMC1laminin, gamma 1 (formerly LAMB2) 0.65 FMOD fibromodulin 0.65 MUC4 mucin4, cell surface associated 0.64 EMID1 EMI domain containing 1 0.62 HMCN1hemicentin 1 0.61 NID1 nidogen 1 0.60 VCAN versican 0.58 CILP2 cartilageintermediate layer protein 2 0.57 SOD3 superoxide dismutase 3,extracellular 0.56 ADAMTS3 ADAM metallopeptidase with thrombospondin0.54 type 1 motif, 3 ZP3 zona pellucida glycoprotein 3 (sperm receptor)0.54 ANGPTL4 angiopoietin-like 4 0.53 CRTAC1 cartilage acidic protein 10.52 LTBP4 latent transforming growth factor beta binding 0.50 protein 4FREM1 FRAS1 related extracellular matrix 1 0.50Table 6 below lists the extracellular matrix genes that correlate withIsl1 expression with a Pearson's correlation of 0.49-0.00.

TABLE 6 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of 0.49 to 0.00 Pearson's Correlationwith Isl1 Gene Extracellular matrix genes (GO: 0031012) Expression SSC5Dscavenger receptor cysteine rich family, 5 0.49 domains GPC6 glypican 60.49 COL1A1 collagen, type I, alpha 1 0.49 ADAMTSL3 ADAMTS-like 3 0.48FLRT3 fibronectin leucine rich transmembrane 0.48 protein 3 FBLN1fibulin 1 0.48 ADAMTS9 ADAM metallopeptidase with 0.48 thrombospondintype 1 motif, 9 COL27A1 collagen, type XXVII, alpha 1 0.47 RELN reelin0.46 COL9A2 collagen, type IX, alpha 2 0.46 EFEMP2 EGF containingfibulin-like extracellular 0.45 matrix protein 2 AGRN agrin 0.44 PCOLCEprocollagen C-endopeptidase enhancer 0.44 NTN4 netrin 4 0.44 CD248 CD248molecule, endosialin 0.44 TGFB1 transforming growth factor, beta 1 0.43ADAMTS2 ADAM metallopeptidase with 0.43 thrombospondin type 1 motif, 2CTHRC1 collagen triple helix repeat containing 1 0.42 FGFR2 fibroblastgrowth factor receptor 2 0.41 APOE apolipoprotein E 0.41 MMP11 matrixmetallopeptidase 11 0.41 MMP15 matrix metallopeptidase 15 (membrane-0.41 inserted) PODN podocan 0.39 VEGFA vascular endothelial growthfactor A 0.39 COL18A1 collagen, type XVIII, alpha 1 0.39 GLG1 golgiglycoprotein 1 0.39 GPC2 glypican 2 0.37 DAG1 dystroglycan 1(dystrophin-associated 0.35 glycoprotein 1) TGFB2 transforming growthfactor, beta 2 0.35 PRELP proline/arginine-rich end leucine-rich 0.35repeat protein CHAD chondroadherin 0.33 COL2A1 collagen, type II, alpha1 0.33 FN1 fibronectin 1 0.31 SMC3 structural maintenance of chromosomes3 0.31 COL4A5 collagen, type IV, alpha 5 0.30 FBN3 fibrillin 3 0.30MMP23B matrix metallopeptidase 23B 0.30 CCDC80 coiled-coil domaincontaining 80 0.29 SPARC secreted protein, acidic, cysteine-rich 0.29(osteonectin) TNXB tenascin XB 0.28 COL6A2 collagen, type VI, alpha 20.28 ADAMTS13 ADAM metallopeptidase with 0.28 thrombospondin type 1motif, 13 LOXL1 lysyl oxidase-like 1 0.28 HAPLN2 hyaluronan andproteoglycan link protein 2 0.28 TNC tenascin C 0.28 ENTPD2ectonucleoside triphosphate 0.28 diphosphohydrolase 2 TGFB3 transforminggrowth factor, beta 3 0.28 MFAP4 microfibrillar-associated protein 40.27 VWF von Willebrand factor 0.27 WNT2 wingless-type MMTV integrationsite family 0.27 member 2 MMRN2 multimerin 2 0.27 SPON1 spondin 1,extracellular matrix protein 0.26 ADAMTS1 ADAM metallopeptidase with0.26 thrombospondin type 1 motif, 1 F2 coagulation factor II (thrombin)0.26 FLRT2 fibronectin leucine rich transmembrane 0.25 protein 2 MMP2matrix metallopeptidase 2 0.24 COL26A1 collagen, type XXVI, alpha 1 0.24CASK calcium/calmodulin-dependent serine protein 0.24 kinase (MAGUKfamily) NTN3 netrin 3 0.23 SLC1A3 solute carrier family 1 (glial highaffinity 0.22 glutamate transporter), member 3 F3 coagulation factor III(thromboplastin, 0.20 tissue factor) ADAMTS6 ADAM metallopeptidase with0.20 thrombospondin type 1 motif, 6 COL5A2 collagen, type V, alpha 20.19 ERBB2IP erbb2 interacting protein 0.18 LAMB1 laminin, beta 1 0.18COLQ collagen-like tail subunit (single strand of 0.18 homotrimer) ofasymmetric acetylcholinesterase LAMA5 laminin, alpha 5 0.17 LOXL2 lysyloxidase-like 2 0.17 WNT11 wingless-type MMTV integration site 0.17family, member 11 LAMB2 laminin, beta 2 (laminin S) 0.17 COL5A1collagen, type V, alpha 1 0.17 AEBP1 AE binding protein 1 0.17 COL9A3collagen, type IX, alpha 3 0.16 CTSD cathepsin D 0.16 COL21A1 collagen,type XXI, alpha 1 0.16 EGFLAM EGF-like, fibronectin type III and laminin0.16 G domains FBN2 fibrillin 2 0.15 NAV2 neuron navigator 2 0.15EMILIN2 elastin microfibril interfacer 2 0.14 WNT9B wingless-type MMTVintegration site 0.14 family, member 9B NOV nephroblastoma overexpressed0.14 CHL1 cell adhesion molecule L1-like 0.13 DLG1 discs, large homolog1 (Drosophila) 0.11 MFGE8 milk fat globule-EGF factor 8 protein 0.11TIMP1 TIMP metallopeptidase inhibitor 1 0.11 CST3 cystatin C 0.10 APLP1amyloid beta (A4) precursor-like protein 1 0.10 PRTN3 proteinase 3 0.10ADAMTS10 ADAM metallopeptidase with 0.09 thrombospondin type 1 motif, 10ILK integrin-linked kinase 0.09 FRAS1 Fraser extracellular matrixcomplex subunit 1 0.09 ANXA2P2 annexin A2 pseudogene 2 0.08 SMOC2 SPARCrelated modular calcium binding 2 0.07 ANXA2 annexin A2 0.06 ODAModontogenic, ameloblast asssociated 0.06 FREM2 FRAS1 relatedextracellular matrix protein 2 0.05 HAPLN3 hyaluronan and proteoglycanlink protein 3 0.05 GPC3 glypican 3 0.03 LGALS1 lectin,galactoside-binding, soluble, 1 0.02 ADAMTS8 ADAM metallopeptidase with0.02 thrombospondin type 1 motif, 8 LUM lumican 0.01 HSP90B1 heat shockprotein 90 kDa beta (Grp94), 0.00 member 1 HAPLN4 hyaluronan andproteoglycan link protein 4 0.00 MATN2 matrilin 2 0.00

Extracellular matrix genes whose expression negatively correlated withIsl1 expression in the HVPs were also identified. Table 7 below liststhe extracellular matrix genes that negatively correlate with Isl1expression with a Pearson's correlation of −0.50 or less.

TABLE 7 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of −0.50 or less Pearson's CorrelationExtracellular matrix genes with Isl1 Gene (GO: 0031012) ExpressionFKBP1A FK506 binding protein 1A, 12 kDa −0.51 CLU clusterin −0.52 TFPI2tissue factor pathway inhibitor 2 −0.52 PLSCR1 phospholipid scramblase 1−0.53 FBLN5 fibulin 5 −0.53 VWA1 von Willebrand factor A domain −0.54containing 1 ADAMTS16 ADAM metallopeptidase with −0.55 thrombospondintype 1 motif, 16 MMP25 matrix metallopeptidase 25 −0.55 SFRP2 secretedfrizzled-related protein 2 −0.61 SOD1 superoxide dismutase 1, soluble−0.68Table 8 below lists the extracellular matrix genes that negativelycorrelate with Isl1 expression with a Pearson's correlation of −0.01 to−0.49.

TABLE 8 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of −0.01 to −0.49 Pearson's Correlationwith Isl1 Gene Extracellular matrix genes (GO: 0031012) Expression PAPLNpapilin, proteoglycan-like sulfated −0.01 glycoprotein SOST sclerostin−0.01 CDON cell adhesion associated, oncogene regulated −0.02 HMCN2hemicentin 2 −0.02 WNT5A wingless-type MMTV integration site family,−0.02 member 5A PCSK6 proprotein convertase subtilisin/kexin type 6−0.02 GSTO1 glutathione S-transferase omega 1 −0.02 LTBP1 latenttransforming growth factor beta −0.03 binding protein 1 KAZALD1Kazal-type serine peptidase inhibitor −0.03 domain 1 LTBP2 latenttransforming growth factor beta −0.03 binding protein 2 SFRP1 secretedfrizzled-related protein 1 −0.04 ADAM11 ADAM metallopeptidase domain 11−0.05 COL6A1 collagen, type VI, alpha 1 −0.05 COL22A1 collagen, typeXXII, alpha 1 −0.05 CYR61 cysteine-rich, angiogenic inducer, 61 −0.05ELN elastin −0.06 COL9A1 collagen, type IX, alpha 1 −0.06 VTNvitronectin −0.06 COL15A1 collagen, type XV, alpha 1 −0.06 COL4A1collagen, type IV, alpha 1 −0.07 ANG angiogenin, ribonuclease, RNase Afamily, 5 −0.07 HSPG2 heparan sulfate proteoglycan 2 −0.09 CRIP2cysteine-rich protein 2 −0.09 CD151 CD151 molecule (Raph blood group)−0.09 THBS1 thrombospondin 1 −0.09 ADAMTS4 ADAM metallopeptidase with−0.09 thrombospondin type 1 motif, 4 CTGF connective tissue growthfactor −0.10 CRISPLD2 cysteine-rich secretory protein LCCL −0.10 domaincontaining 2 BMP7 bone morphogenetic protein 7 −0.11 COL6A3 collagen,type VI, alpha 3 −0.11 COL3A1 collagen, type III, alpha 1 −0.11 COL14A1collagen, type XIV, alpha 1 −0.11 MATN3 matrilin 3 −0.11 CPZcarboxypeptidase Z −0.11 BMP1 bone morphogenetic protein 1 −0.11 WISP1WNT1 inducible signaling pathway −0.12 protein 1 ADAMTS18 ADAMmetallopeptidase with −0.12 thrombospondin type 1 motif, 18 COL7A1collagen, type VII, alpha 1 −0.12 IGFBP7 insulin-like growth factorbinding protein 7 −0.12 COCH cochlin −0.13 ADAMTS5 ADAM metallopeptidasewith −0.13 thrombospondin type 1 motif, 5 COL11A2 collagen, type XI,alpha 2 −0.13 TGFBI transforming growth factor, beta-induced, −0.14 68kDa COL16A1 collagen, type XVI, alpha 1 −0.14 ACHE acetylcholinesterase(Yt blood group) −0.14 THSD4 thrombospondin, type I, domain containing 4−0.15 DGCR6 DiGeorge syndrome critical region gene 6 −0.15 TGFB1I1transforming growth factor beta 1 induced −0.15 transcript 1 ADAMTSL1ADAMTS-like 1 −0.15 SERPINA1 serpin peptidase inhibitor, clade A(alpha-1 −0.16 antiproteinase, antitrypsin), member 1 MAMDC2 MAM domaincontaining 2 −0.16 LAMA4 laminin, alpha 4 −0.17 LTBP3 latenttransforming growth factor beta −0.17 binding protein 3 EGFL7EGF-like-domain, multiple 7 −0.17 NPNT nephronectin −0.17 SERPINF1serpin peptidase inhibitor, clade F (alpha-2 −0.18 antiplasmin, pigmentepithelium derived factor), member 1 ABI3BP ABI family, member 3 (NESH)binding −0.18 protein SERPINE2 serpin peptidase inhibitor, clade E(nexin, −0.18 plasminogen activator inhibitor type 1), member 2 WNT6wingless-type MMTV integration site family, −0.19 member 6 TIMP3 TIMPmetallopeptidase inhibitor 3 −0.19 SNCA synuclein, alpha (non A4component of −0.19 amyloid precursor) PKM pyruvate kinase, muscle −0.19FGF9 fibroblast growth factor 9 −0.19 VIT vitrin −0.19 WNT1wingless-type MMTV integration site family, −0.19 member 1 LAMC3laminin, gamma 3 −0.19 MMP14 matrix metallopeptidase 14 (membrane- −0.19inserted) PXDN peroxidasin −0.19 HNRNPM heterogeneous nuclearribonucleoprotein M −0.19 FBN1 fibrillin 1 −0.20 ASPN asporin −0.20ADAMTSL5 ADAMTS-like 5 −0.20 SPON2 spondin 2, extracellular matrixprotein −0.20 COL1A2 collagen, type I, alpha 2 −0.20 BGN biglycan −0.20COL4A2 collagen, type IV, alpha 2 −0.20 ADAMTSL4 ADAMTS-like 4 −0.21APOH apolipoprotein H (beta-2-glycoprotein I) −0.21 CHI3L1 chitinase3-like 1 (cartilage glycoprotein-39) −0.21 ADAMTS7 ADAM metallopeptidasewith −0.22 thrombospondin type 1 motif, 7 CALR calreticulin −0.22 MMP9matrix metallopeptidase 9 −0.22 MMP24 matrix metallopeptidase 24(membrane- −0.22 inserted) SPOCK2 sparc/osteonectin, cwcv and kazal-like−0.22 domains proteoglycan (testican) 2 COL11A1 collagen, type XI, alpha1 −0.23 MMP7 matrix metallopeptidase 7 −0.23 MMP16 matrixmetallopeptidase 16 (membrane- −0.23 inserted) MFAP2microfibrillar-associated protein 2 −0.23 POSTN periostin, osteoblastspecific factor −0.24 COL8A1 collagen, type VIII, alpha 1 −0.24 WNT2Bwingless-type MMTV integration site family, −0.24 member 2B DCN decorin−0.24 EGFL6 EGF-like-domain, multiple 6 −0.24 MMP10 matrixmetallopeptidase 10 −0.24 MGP matrix Gla protein −0.24 ECM1extracellular matrix protein 1 −0.24 SERPINE1 serpin peptidaseinhibitor, clade E (nexin, −0.25 plasminogen activator inhibitor type1), member 1 MMP1 matrix metallopeptidase 1 −0.25 WNT10A wingless-typeMMTV integration site family, −0.25 member 10A B4GALT7 xylosylproteinbeta 1,4-galactosyltransferase, −0.25 polypeptide 7 COL12A1 collagen,type XII, alpha 1 −0.25 LAMA3 laminin, alpha 3 −0.25 LAMA2 laminin,alpha 2 −0.25 LAMB3 laminin, beta 3 −0.25 WNT7B wingless-type MMTVintegration site family, −0.25 member 7B FLRT1 fibronectin leucine richtransmembrane −0.25 protein 1 ADAMTS15 ADAM metallopeptidase with −0.26thrombospondin type 1 motif, 15 COL8A2 collagen, type VIII, alpha 2−0.26 MFAP1 microfibrillar-associated protein 1 −0.26 TINAGL1tubulointerstitial nephritis antigen-like 1 −0.26 FGF1 fibroblast growthfactor 1 (acidic) −0.27 OLFML2A olfactomedin-like 2A −0.27 CPA6carboxypeptidase A6 −0.27 COL17A1 collagen, type XVII, alpha 1 −0.27SPARCL1 SPARC-like 1 (hevin) −0.27 MFAP5 microfibrillar associatedprotein 5 −0.27 COL4A4 collagen, type IV, alpha 4 −0.28 WNT8Bwingless-type MMTV integration site family, −0.28 member 8B ADAMTS19ADAM metallopeptidase with −0.29 thrombospondin type 1 motif, 19 CRTAPcartilage associated protein −0.29 WNT5B wingless-type MMTV integrationsite family, −0.30 member 5B WNT3 wingless-type MMTV integration sitefamily, −0.30 member 3 UCMA upper zone of growth plate and cartilage−0.30 matrix associated GPC1 glypican 1 −0.30 TIMP2 TIMPmetallopeptidase inhibitor 2 −0.30 ALPL alkaline phosphatase,liver/bone/kidney −0.30 LECT1 leukocyte cell derived chemotaxin 1 −0.32GPC4 glypican 4 −0.32 SPOCK1 sparc/osteonectin, cwcv and kazal-like−0.32 domains proteoglycan (testican) 1 HSD17B12 hydroxysteroid(17-beta) dehydrogenase 12 −0.32 LGALS3 lectin, galactoside-binding,soluble, 3 −0.33 EMILIN3 elastin microfibril interfacer 3 −0.34 GFOD2glucose-fructose oxidoreductase domain −0.34 containing 2 VWC2 vonWillebrand factor C domain containing 2 −0.34 SERAC1 serine active sitecontaining 1 −0.34 WNT8A wingless-type MMTV integration site family,−0.34 member 8A LMCD1 LIM and cysteine-rich domains 1 −0.34 CPXM2carboxypeptidase X (M14 family), member 2 −0.34 ADAMTS14 ADAMmetallopeptidase with −0.34 thrombospondin type 1 motif, 14 GPLD1glycosylphosphatidylinositol specific −0.35 phospholipase D1 FGFBP3fibroblast growth factor binding protein 3 −0.35 BCAN brevican −0.35ITGB4 integrin, beta 4 −0.35 LGALS3BP lectin, galactoside-binding,soluble, −0.36 3 binding protein LPL lipoprotein lipase −0.38 LAD1ladinin 1 −0.39 WNT3A wingless-type MMTV integration site family, −0.39member 3A TGFBR3 transforming growth factor, beta receptor III −0.39 DSTdystonin −0.40 WNT10B wingless-type MMTV integration site family, −0.40member 10B LEFTY2 left-right determination factor 2 −0.41 TNFRSF11Btumor necrosis factor receptor superfamily, −0.41 member 11b WNT9Awingless-type MMTV integration site family, −0.41 member 9A TIMP4 TIMPmetallopeptidase inhibitor 4 −0.42 WNT4 wingless-type MMTV integrationsite family, −0.42 member 4 NCAN neurocan −0.42 ADAMTS20 ADAMmetallopeptidase with −0.43 thrombospondin type 1 motif, 20 ITGA6integrin, alpha 6 −0.43 LOX lysyl oxidase −0.43 THBS4 thrombospondin 4−0.43 THBS2 thrombospondin 2 −0.44 ADAMTSL2 ADAMTS-like 2 −0.44 ENTPD1ectonucleoside triphosphate −0.45 diphosphohydrolase 1 RUNX1runt-related transcription factor 1 −0.45 VWA2 von Willebrand factor Adomain −0.45 containing 2 RELL2 RELT-like 2 −0.46 PTPRZ1 proteintyrosine phosphatase, receptor-type, −0.46 Z polypeptide 1 LAMC2laminin, gamma 2 −0.46 DST dystonin −0.40 WNT10B wingless-type MMTVintegration site family, −0.40 member 10B LEFTY2 left-rightdetermination factor 2 −0.41 TNFRSF11B tumor necrosis factor receptorsuperfamily, −0.41 member 11b WNT9A wingless-type MMTV integration sitefamily, −0.41 member 9A TIMP4 TIMP metallopeptidase inhibitor 4 −0.42

Example 14: Gene Expression Profile for Day 6 Islet 1 Negative Cells

In this example, the gene expression profile was determined for Islet 1negative cells within the Day 6 HVP population to further characterize asubpopulation of cells within the Day 6 population that do not expressthe necessary markers to qualify as engraftable HVPs. Day 6 HVPpopulations were generated as described in Examples 1 or 11 and RNAsequencing (RNA-seq) was performed following differentiation asdescribed in Example 2. Cells that were Islet 1 negative (Isl1−) werefurther analyzed with respect to their gene expression profile. Genesexpressed in the Isl1− cells with an average RNA copy number of 2000 orhigher are shown below in Table 9.

TABLE 9 Gene Expression Profile of Day 6 Islet 1 Negative Cells GeneSample #1 Sample #2 Avg. RNA Copy # ACTB 12288 28126 20207 MTRNR2L224511 9774 17142.5 MALAT1 14163 18092 16127.5 EEF1A1 11663 12456 12059.5KRT8 8884 14087 11485.5 MTRNR2L8 12836 7688 10262 KRT18 5215 105527883.5 FN1 4900 10581 7740.5 MTRNR2L1 8550 5719 7134.5 TTN 3149 96016375 GAPDH 4907 6391 5649 YWHAZ 5349 5414 5381.5 MTRNR2L9 5673 38504761.5 RPL3 3346 5911 4628.5 AHNAK 6727 2197 4462 KCNQ1OT1 5835 30644449.5 TUBB 4870 3936 4403 SLC2A3 3311 4301 3806 FTL 3484 4021 3752.5HSP90B1 4173 2778 3475.5 KRT19 3502 3202 3352 HSPA8 3455 2903 3179 MYL61898 4375 3136.5 RPLP0 2319 3922 3120.5 BSG 2519 3593 3056 COL3A1 5312695 3003.5 TPM1 2938 3059 2998.5 VCAN 2563 3422 2992.5 ENO1 2449 35352992 RPL4 2619 3328 2973.5 ACTG1 2687 3253 2970 MTRNR2L10 3409 2487 2948HMGN2 2684 3153 2918.5 PRTG 2594 2980 2787 TPI1 2418 3113 2765.5 HMGB12577 2880 2728.5 VIM 2621 2704 2662.5 ATP5B 3000 2219 2609.5 HSP90AB12735 2419 2577 RPL7 2132 2896 2514 CBX5 2799 2219 2509 MYL7 1614 33822498 SERPINH1 2547 2327 2437 HNRNPK 2878 1932 2405 SRRM2 2758 2046 2402PODXL 3683 1112 2397.5 EEF2 2579 2119 2349 SPARC 3026 1645 2335.5 ACTC1437 4152 2294.5 HUWE1 2583 1977 2280 COL1A2 3544 941 2242.5 LINC005062965 1496 2230.5 HSPA5 2078 2356 2217 MDK 2223 2144 2183.5 HNRNPC 22922074 2183 HSP90AA1 2220 2138 2179 RGS5 2180 2150 2165 LAMC1 2757 15652161 APLNR 868 3246 2057 UGDH-AS1 2633 1457 2045 RPS3A 1601 2399 2000Accordingly, the data shown in Table 9 provides a gene expressionprofile for Islet 1 negative, non-engraftable cells within a Day 6 HVPpopulation that are not suitable for transplantation and thus are to beselected against when choosing cells for transplantation andengraftment.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A method of transplanting engraftable human ventricular progenitor cells (HVPs), the method comprising: culturing human cells containing cardiac progenitor cells (CPCs) under conditions causing differentiation into human ventricular progenitor cells (HVPs) by subjecting human pluripotent stem cells to activation of Wnt/β-catenin signaling on day 0, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5 to thereby obtain a culture of day 5-7 CPCs comprising day 5-7 LIFR+ Islet1+HVPs; detecting expression on the HVPs of at least one surface marker selected from the group consisting of JAG1, FZD4, FGFR3 and/or TNFSF9 on the day 5-7 HVPs; detecting expression in the day 5-7 HVPs of at least one engraftment markers; isolating day 5-7 HVPs that co-express the at least one surface marker and the at least one engraftment markers to thereby isolate engraftable day 5-7 HVPs; and transplanting the engraftable day 5-7 HVPs into a subject such that the engraftable day 5-7 HVPs form a vascularized, electrically responsive ventricular muscle patch that secretes an extracellular matrix.
 2. The method of claim 1, wherein five or more positive angiogenic markers are detected.
 3. The method of claim 1, wherein ten or more positive angiogenic markers are detected.
 4. The method of claim 1, wherein five or more positive extracellular matrix markers are detected.
 5. The method of claim 1, wherein ten or more positive extracellular matrix markers are detected.
 6. The method of claim 1, wherein detecting expression in the HVPs of at least one engraftment marker comprises detecting expression of at least one positive angiogenic marker and at least one positive extracellular matrix marker.
 7. The method of claim 6, wherein the at least one positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5 and HHEX.
 8. The method of claim 7, wherein the at least one positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21 and HEY1.
 9. The method of claim 7, wherein the at least positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2 and NTRK1.
 10. The method of claim 4, wherein the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 and FREM1.
 11. The method of claim 10, wherein the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF and HTRA1.
 12. The method of claim 10, wherein the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1 and CCBE1.
 13. The method of claim 6, wherein: the at least one positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5 and HHEX; and the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 and FREM1.
 14. The method of claim 13, wherein: the at least one positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21 and HEY1; and the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF and HTRA1.
 15. The method of claim 13, wherein: the at least one positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2 and NTRK1; and the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1 and CCBE1.
 16. The method of claim 1, wherein the at least one engraftment markers detected comprise at least one negative angiogenic marker.
 17. The method of claim 1, wherein the at least one engraftment markers detected comprise at least one negative extracellular matrix marker.
 18. The method of claim 1, herein detecting expression in the HVPs of at least one engraftment marker comprises detecting expression of at least one negative angiogenic marker and at least one negative extracellular matrix marker.
 19. The method of claim 16, wherein the at least one negative angiogenic marker is selected from the group consisting of: ETS1, BAX, XBP1, TDGF1, C5AR1, EPHA1, HS6ST1, SHC1, SP100, JAM3, CASP8, FLT4, SFRP2, HPSE, BAK1, GPX1, VAV3, VAV2, EGF, ADAM15 and AGGF1.
 20. The method of claim 19, wherein the at least one negative angiogenic marker is selected from the group consisting of: VAV3, VAV2, EGF, ADAM15 and AGGF1.
 21. The method of claim 17, wherein the at least one negative extracellular matrix marker is selected from the group consisting of: FKBP1A, CLU, TFP12, PLSCR1, FBLN5, VWA1, ADAMTS16, MMP25, SFRP2 and SOD1.
 22. The method of claim 18, wherein: the at least one negative angiogenic marker is selected from the group consisting of: ETS1, BAX, XBP1, TDGF1, C5AR1, EPHA1, HS6ST1, SHC1, SP100, JAM3, CASP8, FLT4, SFRP2, HPSE, BAK1, GPX1, VAV3, VAV2, EGF, ADAM15 and AGGF1; and the at least one negative extracellular matrix marker is selected from the group consisting of: FKBP1A, CLU, TFP12, PLSCR1, FBLN5, VWA1, ADAMTS16, MMP25, SFRP2 and SOD1.
 23. The method of claim 1, wherein detecting expression of at least one engraftment markers comprises detecting expression of mRNA encoding the at least one engraftment marker in the HVPs.
 24. The method of claim 1, wherein detecting expression of at least one engraftment markers comprises detecting lack of expression of mRNA encoding the at least one engraftment marker in the HVPs.
 25. A method of identifying engraftable human ventricular progenitor cells (HVPs), the method comprising: detecting expression of three or more positive extracellular matrix markers selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 and FREM1 in the HVPs to thereby identify engraftable HVPs, wherein: the HVPs are obtained from a culture of day 5-7 cardiac progenitor cells (CPCs), wherein the culture of day 5-7 CPCs is obtained by subjecting human pluripotent stem cells to activation of the Wnt/β-catenin signaling on day 0, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5; and wherein the HVPs also express at least one surface marker selected from the group consisting of: JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.
 26. The method of claim 25, wherein the three or more positive extracellular matrix markers are selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF and HTRA1.
 27. The method of claim 25, wherein the three or more positive extracellular matrix markers comprise FGF10, SMOC1 and CCBE1.
 28. A method of identifying engraftable human ventricular progenitor cells (HVPs), the method comprising: detecting expression of at least one positive angiogenesis marker selected from the group consisting of PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5 and HHEX; and/or detecting expression of at least one positive extracellular matrix markers selected from the group consisting of SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 and FREM1 in the HVPs to thereby identify engraftable HVPs, wherein: the HVPs are obtained from a culture of day 5-7 cardiac progenitor cells (CPCs), wherein the culture of day 5-7 CPCs is obtained by subjecting human pluripotent stem cells to activation of the Wnt/β-catenin signaling on day 0, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5; and wherein the HVPs also express at least one surface marker selected from the group consisting of: JAG1, FZD4, LIFR and/or TNFSF9.
 29. The method of claim 28, which comprises detecting expression of at least one positive angiogenic marker and at least one positive extracellular matrix marker in the HVPs.
 30. The method of claim 28, wherein the at least one positive angiogenic marker is selected from the group consisting of: PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21 and HEY1.
 31. The method of claim 28, wherein the at least positive angiogenic marker is selected from the group consisting of: PRKD1, CCBE1, PDGFRA, EPHB2, GATA2 and NTRK1.
 32. The method of claim 28, wherein the at least one positive extracellular matrix marker is selected from the group consisting of: SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, FBLN7, FBLN2, NDNF and HTRA1.
 33. The method of claim 28, wherein the at least one positive extracellular matrix marker is selected from the group consisting of: SMOC1 and CCBE1.
 34. A method of identifying engraftable human ventricular progenitor cells (HVPs), the method comprising: detecting expression of ten or more positive angiogenic markers in the HVPs to thereby identify engraftable HVPs, wherein: the HVPs are obtained from a culture of day 5-7 cardiac progenitor cells (CPCs), wherein the culture of day 5-7 CPCs is obtained by subjecting human pluripotent stem cells to activation of the Wnt/β-catenin signaling on day 0, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5; and wherein the HVPs also express at least one surface marker selected from the group consisting of: JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.
 35. The method of claim 34, wherein the ten or more positive angiogenic markers are selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5 and HHEX.
 36. A method of identifying engraftable human ventricular progenitor cells (HVPs), the method comprising: detecting expression of five or more positive extracellular matrix markers in the HVPs to thereby identify engraftable HVPs, wherein: the HVPs are obtained from a culture of day 5-7 cardiac progenitor cells (CPCs), wherein the culture of day 5-7 CPCs is obtained by subjecting human pluripotent stem cells to activation of the Wnt/β-catenin signaling on day 0, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5; and wherein the HVPs also express at least one surface marker selected from the group consisting of: JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.
 37. The method of claim 36, wherein ten or more positive extracellular matrix markers are detected.
 38. The method of claim 36, wherein the five or more positive extracellular matrix marker are selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 and FREM1. 